U.S. patent application number 10/318608 was filed with the patent office on 2003-07-10 for il-13 receptor specific chimeric proteins & uses thereof.
This patent application is currently assigned to The Government of the USA as represented by the Secretary of the Dept. of Health & Human Services. Invention is credited to Debinski, Waldemar, Obiri, Nicholas, Pastan, Ira, Puri, Raj K..
Application Number | 20030129132 10/318608 |
Document ID | / |
Family ID | 25433211 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129132 |
Kind Code |
A1 |
Puri, Raj K. ; et
al. |
July 10, 2003 |
IL-13 receptor specific chimeric proteins & uses thereof
Abstract
The present invention provides a method and compositions for
specifically delivering an effector molecule to a tumor cell. The
method involves providing a chimeric molecule that comprises an
effector molecule attached to a targeting molecule that
specifically binds an IL-13 receptor and contacting a tumor cell
with the chimeric molecule.
Inventors: |
Puri, Raj K.; (North
Potomac, MD) ; Debinski, Waldemar; (Hershey, PA)
; Pastan, Ira; (Potomac, MD) ; Obiri,
Nicholas; (N. Potomac, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Government of the USA as
represented by the Secretary of the Dept. of Health & Human
Services
|
Family ID: |
25433211 |
Appl. No.: |
10/318608 |
Filed: |
December 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10318608 |
Dec 13, 2002 |
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08913370 |
Feb 17, 1998 |
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6518061 |
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08913370 |
Feb 17, 1998 |
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PCT/US96/03486 |
Mar 15, 1996 |
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08913370 |
Feb 17, 1998 |
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08404685 |
Mar 15, 1995 |
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5614191 |
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Current U.S.
Class: |
424/1.49 ;
424/145.1; 424/178.1; 424/450; 424/85.2 |
Current CPC
Class: |
C07K 16/2866 20130101;
A61K 47/642 20170801; C07K 2319/00 20130101; A61K 47/6849 20170801;
A61K 2039/505 20130101 |
Class at
Publication: |
424/1.49 ;
424/85.2; 424/178.1; 424/145.1; 424/450 |
International
Class: |
A61K 051/00; A61K
039/395; A61K 038/20; A61K 009/127 |
Claims
What is claimed is:
1. A method for specifically delivering an effector molecule to a
tumor cell bearing an IL-13 receptor, said method comprising:
providing a chimeric molecule comprising said effector molecule
attached to a targeting molecule that specifically binds to an
IL-13 receptor; and contacting said tumor with said chimeric
molecule; wherein said chimeric molecule specifically binds to a
tumor cell.
2. The method of claim 1, wherein said targeting molecule is
IL-13.
3. The method of claim 1, wherein said targeting molecule is an
anti-IL-13 receptor antibody.
4. The method of claim 1, wherein said targeting molecule is a
circularly permuted IL-13.
5. The method of claim 1, wherein said tumor is selected from the
group consisting of a carcinoma.
6. The method of claim 1, wherein said tumor is selected from the
group consisting of a renal cell carcinoma, a glioma, a
medulloblastoma, a renal cell carcinoma, and a Kaposi's
sarcoma.
7. The method of claim 1, wherein said effector molecule is
selected from the group consisting of a cytotoxin, a label, a
radionuclide, a drug, a liposome, a ligand, and an antibody.
8. The method of claim 7, wherein said effector molecule is a
Pseudomonas exotoxin.
9. The method of claim 8, wherein chimeric molecule is a fusion
protein.
10. The method of claim 9, wherein said a fusion protein is
IL-13-PE38QQR.
11. The method of claim 9, wherein said a fusion protein is
cpIL-13-PE4E.
12. The method of claim 9, wherein said a fusion protein is
IL-13-PE4E.
13. The method of claim 9, wherein said a fusion protein is
cpIL-13-PE4E.
14. A method for impairing growth of tumor cells bearing an IL-13
receptor, said method comprising contacting said tumor with a
chimeric molecule comprising: a targeting molecule that
specifically binds a human IL-13 receptor; and an effector molecule
selected from the group consisting of a cytotoxin, a radionuclide,
a ligand and an antibody; wherein said chimeric molecule
specifically binds to a tumor cell.
15. The method of claim 14, wherein said targeting molecule is an
antibody that specifically binds a human IL-13 receptor.
16. The method of claim 14, wherein said targeting molecule is a
human IL-13.
17. The method of claim 14, wherein said targeting molecule is a
circularly permuted human IL-13.
18. The method of claim 16, 17, wherein said effector molecule is a
cytotoxin.
19. The method of claim 18, wherein said cytotoxin is selected from
the group consisting of Pseudomonas exotoxin, ricin, abrin and
Diphtheria toxin.
20. The method of claim 19, wherein chimeric molecule is a
single-chain fusion protein.
21. The method of claim 19, wherein said cytotoxin is a Pseudomonas
exotoxin.
22. The method of claim 21, wherein said Pseudomonas exotoxin is
PE38QQR.
23. The method of claim 21, wherein said Pseudomonas exotoxin is
PE4E.
24. The method of claim 16, 17, wherein said tumor cell growth is
tumor cell growth in a human.
25. The method of claim 24, wherein said contacting comprises
administering said chimeric molecule to the human intravenously,
into a body cavity, or into a lumen or an organ.
26. A method for detecting the presence or absence of a tumor, said
method comprising contacting said tumor with a chimeric molecule
comprising: a targeting molecule that specifically binds a human
IL-13 receptor; and a detectable label; and detecting the presence
or absence of said label.
27. A vector comprising a nucleic acid sequence encoding a chimeric
fusion protein comprising an IL-13 or circularly permuted IL-13
attached to a polypeptide wherein said chimeric fusion protein
specifically binds to a tumor cell bearing an IL-13 receptor.
28. The vector of claim 27, wherein said nucleic acid sequence
encodes an IL-13-PE fusion protein.
29. The vector of claim 27, wherein said nucleic acid sequence
encodes a cpIL-13-PE fusion protein.
30. The vector of claim 28, 29, wherein said nucleic acid sequence
encodes a fusion protein selected from the group consisting of
IL-13-PE38QQR, cpIL-13-PE38QQR, IL-13-PE4E, and cpIL-13-PE4E.
31. A host cell comprising a nucleic acid sequence encoding a
chimeric fusion protein comprising an IL-13 or a circularly
permuted IL-13 attached to a polypeptide wherein said chimeric
fusion protein specifically binds to a tumor cell bearing an IL-13
receptor.
32. The host cell of claim 31, wherein said nucleic acid sequence
encodes an IL-13-PE fusion protein.
33. The vector of claim 32, wherein said nucleic acid sequence
encodes a fusion protein selected from the group consisting of
IL-13-PE38QQR, cpIL-13-PE38QQR, IL-13-PE4E, and cpIL-13-PE4E.
34. A chimeric molecule that specifically binds a tumor cell
bearing an IL-13 receptor, said chimeric molecule comprising a
cytotoxic molecule attached to a targeting molecule that
specifically binds an IL-13 receptor.
35. The composition of claim 34, 34, wherein said targeting
molecule is human IL-13.
36. The composition of claim 34, 34, wherein said cytotoxin is
selected from the group consisting of Pseudomonas exotoxin, ricin,
abrin and Diphtheria toxin.
37. The composition of claim 35, wherein chimeric molecule is a
single-chain fusion protein.
38. The method of claim 37, wherein said cytotoxin is a Pseudomonas
exotoxin.
39. The composition of claim 38, 39, 41, 46, wherein said
Pseudomonas exotoxin is PE38QQR or PE4E.
40. A chimeric molecule that specifically binds a tumor cell
bearing an IL-13 receptor, said chimeric molecule comprising an
effector molecule attached to an antibody that specifically binds
an IL-13 receptor.
41. The composition of claim 40, wherein said effector molecule is
selected from the group consisting of a cytotoxin, a label, a
radionuclide, a drug, a liposome, a ligand, and an antibody.
42. A pharmacological composition comprising a pharmaceutically
acceptable carrier and a chimeric molecule, said chimeric molecule
comprising: an effector molecule attached to a targeting molecule
that specifically binds to an IL-13 receptor.
43. The composition of claim 42, wherein said targeting molecule is
selected from the group consisting of IL-13, and circularly
permuted IL-13.
44. The composition of claim 43, wherein said effector molecule is
selected from the group consisting of a cytotoxin, a label, a
radionuclide, a drug, a liposome, a ligand, and an antibody.
45. The composition of claim 44, wherein chimeric molecule is a
single-chain fusion protein.
46. The composition of claim 45, wherein said Pseudomonas exotoxin
is PE38QQR or PE4E.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/404,685 filed Mar. 15, 1995 which is
incorporated herein by reference for all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to methods of specifically delivering
an effector molecule to a tumor cell. In particular this invention
relates to chimeric molecules that specifically bind to IL-13
receptors and their use to deliver molecules having a particular
activity to tumors overexpressing IL-13 receptors.
BACKGROUND OF THE INVENTION
[0003] In a chimeric molecule, two or more molecules that exist
separately in their native state are joined together to form a
single molecule having the desired functionality of all of its
constituent molecules. Frequently, one of the constituent molecules
of a chimeric molecule is a "targeting molecule". The targeting
molecule is a molecule such as a ligand or an antibody that
specifically binds to its corresponding target, for example a
receptor on a cell surface. Thus, for example, where the targeting
molecule is an antibody, the chimeric molecule will specifically
bind (target) cells and tissues bearing the epitope to which the
antibody is directed.
[0004] Another constituent of the chimeric molecule may be an
"effector molecule". The effector molecule refers to a molecule
that is to be specifically transported to the target to which the
chimeric molecule is specifically directed. The effector molecule
typically has a characteristic activity that is desired to be
delivered to the target cell. Effector molecules include
cytotoxins, labels, radionuclides, ligands, antibodies, drugs,
liposomes, and the like.
[0005] In particular, where the effector component is a cytotoxin,
the chimeric molecule may act as a potent cell-killing agent
specifically targeting the cytotoxin to cells bearing a particular
target molecule. For example, chimeric fusion proteins which
include interleukin 4 (IL-4) or transforming growth factor
(TGF.alpha.) fused to Pseudomonas exotoxin (PE) or interleukin 2
(IL-2) fused to Diphtheria toxin (DT) have been shown to
specifically target and kill cancer cells (Pastan et al., Ann. Rev.
Biochem., 61: 331-354 (1992)).
[0006] Generally, it is desirable to increase specificity and
affinity and decrease cross-reactivity of chimeric cytotoxins in
order to increase their efficacy. To the extent a chimeric molecule
preferentially selects and binds to its target (e.g. a tumor cell)
and not to a non-target (e.g. a healthy cell), side effects of the
chimeric molecule will be minimized. Unfortunately, many targets to
which chimeric cytotoxins have been directed (e.g. the IL-2
receptor), while showing elevated expression on tumor cells, are
also expressed at significant levels on healthy cells. Thus,
chimeric cytotoxins directed to these targets frequently show
adverse side-effects as they bind non-target (e.g., healthy) cells
that also express the targeted receptor.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods and compositions for
specifically delivering an effector molecule to a tumor cell. In
particular, the present invention provides chimeric molecules that
specifically target tumor cells with less binding to healthy cells
than other analogous chimeric molecules known in the prior art.
[0008] The improved specific targeting of this invention is
premised, in part, on the discovery that tumor cells, especially
carcinomas such as renal cell carcinoma, overexpress IL-13
receptors at extremely high levels. The extremely high level of
IL-13 receptor expression on target tumor cells permits the use of
lower dosages of chimeric molecule to deliver the same amount of
effector molecule to the target cells and also results in reduced
binding of non-tumor cells.
[0009] In a preferred embodiment, this invention provides for a
method for specifically delivering an effector molecule to a tumor
cell bearing an IL-13 receptor. The method involves providing a
chimeric molecule comprising an effector molecule attached to a
targeting molecule that specifically binds to an IL-13 receptor and
contacting the tumor with the chimeric molecule resulting in
binding of the chimeric molecule to the tumor cell.
[0010] The targeting molecule is preferably either a ligand, such
as IL-13 or circularly permuted IL-13 (cpIL-13, especially cpIL-13
where the native IL-13 is opened between residues 43 and 44 (Gly
and Met respectively) to produce a cpIL-13 having Met44 as the
amino terminus and Gly43 as its carboxyl terminus) that
specifically binds an IL-13 receptor, or an anti-IL-13 receptor
antibody. The targeting molecule may be conjugated to the effector
molecule, or where both targeting and effector molecules are
polypeptides, the targeting molecule may be joined to the effector
molecule through one or more peptide bonds thereby forming a fusion
protein. Suitable effector molecules include a cytotoxin, a label,
a radionuclide, a drug, a liposome, a ligand, and an antibody. In a
particularly preferred embodiment, the effector is a cytotoxin,
more specifically a Pseudomonas exotoxin such as PE38QQR or PE4E.
Where the Pseudomonas exotoxin is fused to an IL-13 targeting
molecule, preferred fusion proteins include, but are not limited to
IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, and cpIL-13-PE4E.
[0011] In another embodiment, this invention provides a method for
impairing the growth of tumor cells, more preferably solid tumor
cells, bearing an IL-13 receptor. The method involves contacting
the tumor with a chimeric molecule comprising an effector molecule
selected from the group consisting of a cytotoxin, a radionuclide,
a ligand and an antibody. The effector molecule is attached to a
targeting molecule that specifically binds a human IL-13 receptor.
The targeting molecule is preferably a ligand (such as IL-13) that
binds the IL-13 receptor or an anti-IL-13 receptor antibody.
Preferred cytotoxic effector molecules include Pseudomonas
exotoxin, Diphtheria toxin, ricin and abrin. Psuedomonas exotoxins,
such as PE38QQR and PE4E, are particularly preferred. The targeting
molecule may be conjugated or fused to the effector molecule with
attachment by fusion preferred for cytotoxic effector molecules.
The tumor growth that is impaired may be tumor growth in a human.
Thus the method may further comprise administering the chimeric
molecule to a human intravenously into a body cavity, or into a
human or an organ.
[0012] In yet another embodiment, this invention provides for a
method of detecting the presence or absence of a tumor. The method
involves contacting the tumor with a chimeric molecule comprising a
detectable label attached to a targeting molecule that specifically
binds a human IL-13 receptor and detecting the presence or absence
of the label. In a preferred embodiment, the label is selected from
the group consisting of a radioactive label, an enzymatic label, an
electron dense label, and a fluorescent label. Preferred targeting
molecules include, but are not limited to IL-13, cpIL-13, and
anti-IL-13R antibodies.
[0013] This invention also provides for vectors comprising a
nucleic acid sequence encoding a chimeric polypeptide fusion
protein comprising an IL-13, or a cpIL-13, attached to a second
polypeptide. The chimeric polypeptide fusion protein specifically
binds to a tumor cell bearing an IL-13 receptor. A preferred vector
encodes an IL-13-PE or cpIL-13-PE fusion protein and more
preferably encodes an IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR,
or cpIL-13-PE4E fusion protein.
[0014] This invention also provides for host cells comprising a
nucleic acid sequence encoding a chimeric polypeptide fusion
protein comprising an IL-13 attached to a second polypeptide. A
preferred host cell comprises a nucleic acid encoding an IL-13-PE,
or cpIL-13-PE, fusion protein, more preferably encoding an
IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, or cpIL-13-PE4E fusion
protein. The encoded fusion protein specifically binds to a tumor
cell bearing an IL-13 receptor. Particularly preferred host cells
are bacterial host cells, especially E. coli cells.
[0015] In still yet another embodiment, this invention provides
chimeric molecules that specifically bind a tumor cell bearing an
IL-13 receptor. In one preferred embodiment, the chimeric molecule
comprises a cytotoxic molecule attached to a targeting molecule
that specifically binds an IL-13. The targeting molecule may be
conjugated or fused to the cytotoxic molecule. In a preferred
embodiment, the targeting molecule is fused to the cytotoxin
thereby forming a single-chain fusion protein. Particularly
preferred targeting molecules are IL-13, cpIL-13, or an antibody
that specifically binds to the IL-13 receptor. Preferred cytotoxic
molecules include Pseudomonas exotoxin, Diphtheria toxin, ricin,
and abrin, with Pseudomonas exotoxins (especially PE38QQR or PE4E)
being most preferred.
[0016] In another preferred embodiment, the chimeric molecule
comprises an effector molecule attached to an antibody that
specifically binds to an IL-13 receptor. Effector molecules include
a cytotoxin, a label, a radionuclide, a drug, liposome, a ligand
and an antibody. The effector molecule may be fused or conjugated
to the antibody.
[0017] The invention additionally provides for pharmacological
compositions comprising a pharmaceutically acceptable carrier and a
chimeric molecule where the chimeric molecule comprises and
effector molecule attached to a targeting molecule that
specifically binds to an IL-13 receptor. The targeting and effector
molecules may be conjugated or fused to each other. Particularly
preferred targeting molecules include IL-13, cpIL-13, and
anti-IL-13 receptor antibodies, while preferred effector molecules
include a cytotoxin, a label, a radionuclide, a drug, a liposome, a
ligand and an antibody. A preferred pharmacological composition
includes an IL-13-PE fusion protein, more preferably a
IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, or cpIL-13-PE4E fusion
protein.
[0018] Definitions
[0019] The term "specifically deliver" as used herein refers to the
preferential association of a molecule with a cell or tissue
bearing a particular target molecule or marker and not to cells or
tissues lacking that target molecule. It is, of course, recognized
that a certain degree of non-specific interaction may occur between
a molecule and a non-target cell or tissue. Nevertheless, specific
delivery, may be distinguished as mediated through specific
recognition of the target molecule. Typically specific delivery
results in a much stronger association between the delivered
molecule and cells bearing the target molecule than between the
delivered molecule and cells lacking the target molecule. Specific
delivery typically results in greater than 2 fold, preferably
greater than 5 fold, more preferably greater than 10 fold and most
preferably greater than 100 fold increase in amount of delivered
molecule (per unit time) to a cell or tissue bearing the target
molecule as compared to a cell or tissue lacking the target
molecule or marker.
[0020] The term "residue" as used herein refers to an amino acid
that is incorporated into a polypeptide. The amino acid may be a
naturally occurring amino acid and, unless otherwise limited, may
encompass known analogs of natural amino acids that can function in
a similar manner as naturally occurring amino acids.
[0021] A "fusion protein" refers to a polypeptide formed by the
joining of two or more polypeptides through a peptide bond formed
between the amino terminus of one polypeptide and the carboxyl
terminus of another polypeptide. The fusion protein may be formed
by the chemical coupling of the constituent polypeptides or it may
be expressed as a single polypeptide from nucleic acid sequence
encoding the single contiguous fusion protein. A single chain
fusion protein is a fusion protein having a single contiguous
polypeptide backbone.
[0022] A "spacer" as used herein refers to a peptide that joins the
proteins comprising a fusion protein. Generally a spacer has no
specific biological activity other than to join the proteins or to
preserve some minimum distance or other spatial relationship
between them. However, the constituent amino acids of a spacer may
be selected to influence some property of the molecule such as the
folding, net charge, or hydrophobicity of the molecule.
[0023] A "ligand", as used herein, refers generally to all
molecules capable of reacting with or otherwise recognizing or
binding to a receptor on a target cell. Specifically, examples of
ligands include, but are not limited to, antibodies, lymphokines,
cytokines, receptor proteins such as CD4 and CD8, solubilized
receptor proteins such as soluble CD4, hormones, growth factors,
and the like which specifically bind desired target cells.
[0024] The term "cpIL-13" is used to designate a circularly
permuted (cp) IL-13. Circular permutation is functionally
equivalent to taking a straight-chain molecule, fusing the ends
(directly or through a linker) to form a circular molecule, and
then cutting the circular molecule at a different location to form
a new straight chain molecule with different termini
DETAILED DESCRIPTION
[0025] I. Chimeric Molecules Targeted to the IL-13 Receptor.
[0026] The present invention provides a method for specifically
delivering an effector molecule to a tumor cell. This method
involves the use of chimeric molecules comprising a targeting
molecule attached to an effector molecule. The chimeric molecules
of this invention specifically target tumor cells while providing
reduced binding to non-target cells as compared to other targeted
chimeric molecules known in the art.
[0027] The improved specific targeting of this invention is
premised, in part, on the discovery that solid tumors, especially
carcinomas, overexpress IL-13 receptors at extremely high levels.
While the IL-13 receptors (IL-13R) are overexpressed on tumor
cells, expression on other cells (e.g. monocytes, B cells, and T
cells) appears negligible. Thus, by specifically targeting the
IL-13 receptor, the present invention provides chimeric molecules
that are specifically directed to solid tumors while minimizing
targeting of other cells or tissues.
[0028] In a preferred embodiment, this invention provides for
compositions and methods for impairing the growth of tumors. The
methods involve providing a chimeric molecule comprising a
cytotoxic effector molecule attached to a targeting molecule that
specifically binds an IL-13 receptor. The cytotoxin may be a native
or modified cytotoxin such as Pseudomonas exotoxin (PE), Diphtheria
toxin (DT), ricin, abrin, and the like.
[0029] The chimeric cytotoxin is administered to an organism
containing tumor cells which are then contacted by the chimeric
molecule. The targeting molecule component of the chimeric molecule
specifically binds to the overexpressed IL-13 receptors on the
tumor cells. Once bound to the IL-13 receptor on the cell surface,
the cytotoxic effector molecule mediates internalization into the
cell where the cytotoxin inhibits cellular growth or kills the
cell.
[0030] The use of chimeric molecules comprising a targeting moiety
joined to a cytotoxic effector molecules to target and kill tumor
cells is known in the prior art. For example, chimeric fusion
proteins which include interleukin 4 (IL-4) or transforming growth
factor (TGF.alpha.) fused to Pseudomonas exotoxin (PE) or
interleukin 2 (1L-2) fused to Diphtheria toxin (DT) have been
tested for their ability to specifically target and kill cancer
cells (Pastan et al., Ann. Rev. Biochem., 61: 331-354 (1992)).
[0031] Although chimeric IL-4-cytotoxin molecules are known in the
prior art, and IL-4 shows some sequence similarity to IL-13, it was
an unexpected discovery of the present invention that cytotoxins
targeted by a moiety specific to the IL-13 receptor show
significantly increased efficacy as compared to IL-4 receptor
directed cytotoxins. Without being bound to a particular theory, it
is believed that the improved efficacy of the IL-13 chimeras of the
present invention is due to at least three factors.
[0032] First, IL-13 receptors are expressed at much lower levels,
if at all on non-tumor cells (e.g. monocytes, T cells, B cells).
Thus cytotoxins directed to IL-13 receptors show reduced binding
and subsequent killing of healthy cells and tissues as compared to
other cytotoxins.
[0033] Second, the receptor component that specifically binds IL-13
appears to be expressed at significantly higher levels on solid
tumors than the receptor component that binds IL-4. Thus, tumor
cells bind higher levels of cytotoxic chimeric molecules directed
against IL-13 receptors than cytotoxic chimeric molecules directed
against IL-4 receptors.
[0034] Finally, IL-4 receptors are up-regulated when immune system
cells (e.g. T-cells) are activated. This results in healthy cells,
for example T-cells and B-cells, showing greater susceptibility to
IL-4 receptor directed cytotoxins. Thus, the induction of an immune
response (as against a cancer), results in greater susceptibility
of cells of the immune system to the therapeutic agent. In
contrast, IL-13 receptors have not been shown to be up-regulated in
activated T cells. Thus IL-13 receptor targeted cytotoxins have no
greater effect on activated T cells and thereby minimize adverse
effects of the therapeutic composition on cells of the immune
system.
[0035] In another embodiment, this invention also provides for
compositions and methods for detecting the presence or absence of
tumor cells. These methods involve providing a chimeric molecule
comprising an effector molecule, that is a detectable label
attached to a targeting molecule that specifically binds an IL-13
receptor. The IL-13 receptor targeting moiety specifically binds
the chimeric molecule to tumor cells which are then marked by their
association with the detectable label. Subsequent detection of the
cell-associated label indicates the presence of a tumor cell.
[0036] In yet another embodiment, the effector molecule may be
another specific binding moiety such as an antibody, a growth
factor, or a ligand. The chimeric molecule will then act as a
highly specific bifunctional linker. This linker may act to bind
and enhance the interaction between cells or cellular components to
which the fusion protein binds. Thus, for example, where the
"targeting" component of the chimeric molecule comprises a
polypeptide that specifically binds to an IL-13 receptor and the
"effector" component is an antibody or antibody fragment (e.g. an
Fv fragment of an antibody), the targeting component specifically
binds cancer cells, while the effector component binds receptors
(e.g., IL-2 or IL-4 receptors) on the surface of immune cells. The
chimeric molecule may thus act to enhance and direct an immune
response toward target cancer cells.
[0037] In still yet another embodiment the effector molecule may be
a pharmacological agent (e.g. a drug) or a vehicle containing a
pharmacological agent. This is particularly suitable where it is
merely desired to invoke a non-lethal biological response. Thus the
moiety that specifically binds to an IL-13 receptor may be
conjugated to a drug such as vinblastine, doxirubicin, genistein (a
tyrosine kinase inhibitor), an antisense molecule, and other
pharmacological agents known to those of skill in the art, thereby
specifically targeting the pharmacological agent to tumor cells
over expressing IL-13 receptors.
[0038] Alternatively, the targeting molecule may be bound to a
vehicle containing the therapeutic composition. Such vehicles
include, but are not limited to liposomes, micelles, various
synthetic beads, and the like.
[0039] One of skill in the art will appreciate that the chimeric
molecules of the present invention may include multiple targeting
moieties bound to a single effector or conversely, multiple
effector molecules bound to a single targeting moiety. In still
other embodiment, the chimeric molecules may include both multiple
targeting moieties and multiple effector molecules. Thus, for
example, this invention provides for "dual targeted" cytotoxic
chimeric molecules in which targeting molecule that specifically
binds to IL-13 is attached to a cytotoxic molecule and another
molecule (e.g. an antibody, or another ligand) is attached to the
other terminus of the toxin. Such a dual-targeted cytotoxin might
comprise an IL-13 substituted for domain Ia at the amino terminus
of a PE and anti-TAC(Fv) inserted in domain III, between amino acid
604 and 609. Other antibodies may also be suitable.
[0040] II. The Targeting Molecule.
[0041] In a preferred embodiment, the targeting molecule is a
molecule that specifically binds to the IL-13 receptor. The term
"specifically binds", as used herein, when referring to a protein
or polypeptide, refers to a binding reaction which is determinative
of the presence of the protein or polypeptide in a heterogeneous
population of proteins and other biologics. Thus, under designated
conditions (e.g. immunoassay conditions in the case of an
antibody), the specified ligand or antibody binds to its particular
"target" protein (e.g. an IL-13 receptor protein) and does not bind
in a significant amount to other proteins present in the sample or
to other proteins to which the ligand or antibody may come in
contact in an organism.
[0042] A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with an IL-13 receptor
protein. For example, solid-phase ELISA immunoassays are routinely
used to select monoclonal antibodies specifically immunoreactive
with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0043] Similarly, assay formats for detecting specific binding of
ligands (e.g. IL-13, cpIL-13) with their respective receptors are
also well known in the art. Example 1 provides a detailed protocol
for assessing specific binding of labeled IL-13 by an IL-13
receptor.
[0044] The IL-13 receptor is a cell surface receptor that
specifically binds IL-13 and mediates a variety of physiological
responses in various cell types as described below in the
description of IL-13. The IL-13 receptor may be identified by
contacting a cell or other sample with labeled IL-13 and detecting
the amount of specific binding of IL-13 according to methods well
known to those of skill in the art. Detection of IL-13 receptors by
labeled IL-13 binding is described in detail in Example 1.
[0045] Alternatively, an anti-IL-13 receptor antibody may also be
used to identify IL-13 receptors. The antibody will specifically
bind to the IL-13 receptor and this binding may be detected either
through detection of a conjugated label or through detection of a
labeled second antibody that binds the anti-IL-13 receptor
antibody.
[0046] In a preferred embodiment, the moiety utilized to
specifically target the IL-13 receptor is either an antibody that
specifically binds the IL-13 receptor (an anti-IL-13R antibody) or
a ligand, such as IL-13 or cpIL-13, that specifically binds to the
receptor.
[0047] A) IL-13.
[0048] Interleukin-13 (IL-13) is a pleiotropic cytokine that is
recognized to share many of the properties of IL-4. IL-13 has
approximately 30% sequence identity with IL-4 and exhibits
IL-4-like activities on monocytes/macrophages and human B cells
(Minty et al., Nature, 362: 248 (1993), McKenzie et al. Proc. Natl.
Acad. Sci. USA, 90: 3735 (1987)). In particular, IL-13 appears to
be a potent regulator of inflammatory and immune responses. Like
IL-4, IL-13 can up-regulate the monocyte/macrophage expression of
CD23 and MHC class I and class II antigens, down-regulate the
expression of Fc.gamma., and inhibit antibody-dependent
cytotoxicity. IL-13 can also inhibit nitric oxide production as
well as the expression of pro-inflammatory cytokines (e.g. IL-1,
IL-6, IL-8, IL-10 and IL-12) and chemokines (MIP-1, MCP), but
enhance the production of IL-1 (Minty supra.; Mckenzie et al.,
supra.; Zurawski et al. Immunol. Today, 15: 19 (1994); de Wall
Malefyt et al. J. Immunol., 150: 180A (1993); de Wall Malefyt et
al. J. Immunol., 151: 6370 (1993); Doherty et al. J. Immunol., 151:
7151 (1993); and Minty et al. Eur. cytokine Netw., 4: 99
(1993)).
[0049] Recombinant IL-13 is commercially available from a number of
sources (see, e.g. R & D Systems, Minneapolis, Minn., USA, and
Sanofi Bio-Industries, Inc., Tervose, Pa., USA). Alternatively, a
gene or a cDNA encoding IL-13 may be cloned into a plasmid or other
expression vector and expressed in any of a number of expression
systems according to methods well known to those of skill in the
art. Methods of cloning and expressing IL-13 and the nucleic acid
sequence for IL-13 are well known (see, for example, Minty et al.
(1993) supra. and McKenzie (1987), supra). In addition, the
expression of IL-13 as a component of a chimeric molecule is
detailed in Example 4.
[0050] One of skill in the art will appreciate that analogues or
fragments of IL-13 bearing will also specifically bind to the IL-13
receptor. For example, conservative substitutions of residues
(e.g., a serine for an alanine or an aspartic acid for a glutamic
acid) comprising native IL-13 will provide IL-13 analogues that
also specifically bind to the IL-13 receptor. Thus, the term
"IL-13", when used in reference to a targeting molecule, also
includes fragments, analogues or peptide mimetics of IL-13 that
also specifically bind to the IL-13 receptor.
[0051] B) Anti-IL-13 Receptor Antibodies.
[0052] i) The Antibodies.
[0053] One of skill will recognize that other molecules besides
IL-13 will specifically bind to IL-13 receptors. Polyclonal and
monoclonal antibodies directed against IL-13 receptors provide
particularly suitable targeting molecules in the chimeric molecules
of this invention. The term "antibody", as used herein, includes
various forms of modified or altered antibodies, such as an intact
immunoglobulin, various fragments such as an Fv fragment, an Fv
fragment containing only the light and heavy chain variable
regions, an Fv fragment linked by a disulfide bond (Brinkmann, et
al. Proc. Natl. Acad. Sci. USA, 90: 547-551 (1993)), an Fab or
(Fab)'.sub.2 fragment containing the variable regions and parts of
the constant regions, a single-chain antibody and the like (Bird et
al., Science 242: 424-426 (1988); Huston et al., Proc. Nat. Acad.
Sci. USA 85: 5879-5883 (1988)). The antibody may be of animal
(especially mouse or rat) or human origin or may be chimeric
(Morrison et al., Proc Nat. Acad. Sci. USA 81: 6851-6855 (1984)) or
humanized (Jones et al., Nature 321: 522-525 (1986), and published
UK patent application #8707252). Methods of producing antibodies
suitable for use in the present invention are well known to those
skilled in the art and can be found described in such publications
as Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988), and Asai, Methods in Cell Biology Vol.
37: Antibodies in Cell Biology, Academic Press, Inc. N.Y.
(1993).
[0054] Antibodies that specifically bind the IL-13 receptor may be
produced by a number of means well known to those of skill in the
art. Generally, this involves using an antigenic component of the
IL-13 receptor as an antigen to induce the production of antibodies
in an organism (e.g. a sheep, mouse, rabbit, etc.). One of skill in
the art will recognize that there are numerous methods of isolating
all or components of the IL-13 receptor for use as an antigen. For
example, IL-13 receptors may be isolated by cross-linking the
receptor to a labeled IL-13 by the exposure to 2 mM disuccinimidyl
suberate (DSS). The labeled receptor may then be isolated according
to routine methods and the isolated receptor may be used as an
antigen to raise anti-IL-13 receptor antibodies as described below.
Cross-linking and isolation of components of the IL-13 receptor is
described in Example 3.
[0055] In a preferred embodiment, however, IL-13 receptors may be
isolated by means of affinity chromatography. It was a surprising
discovery of the present invention that solid tumor cells
overexpress IL-13 receptors. This discovery of cells overexpressing
IL-13 receptor greatly simplifies the receptor isolation.
Generally, approximately, 100 million renal carcinoma cells, may be
solubilized in detergent with protease inhibitors according to
standard methods. The resulting lysate is then run through an
affinity column bearing IL-13. The receptor binds to the IL-13 in
the column thereby effecting an isolation from the lysate. The
column is then eluted with a low pH buffer to dissociate the IL-13
ligand from the IL-13 receptor resulting in isolated receptor. The
isolated receptor may then be used as an antigen to raise
anti-IL-13 receptor antibodies.
[0056] ii) Antibody Production.
[0057] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably an
isolated IL-13 receptor or receptor epitope is mixed with an
adjuvant and animals are immunized with the mixture. The animal's
immune response to the immunogen preparation is monitored by taking
test bleeds and determining the titer of reactivity to the
polypeptide of interest. When appropriately high titers of antibody
to the immunogen are obtained, blood is collected from the animal
and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the polypeptide is performed
where desired. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies:
A Laboratory Manual Cold Spring Harbor Press, NY).
[0058] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Description of techniques for
preparing such monoclonal antibodies may be found in, e.g., Stites
et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y.; and particularly in Kohler and
Milstein (1975) Nature 256: 495-497, which discusses one method of
generating monoclonal antibodies.
[0059] Summarized briefly, this method involves injecting an animal
with an immunogen. The animal is then sacrificed and cells taken
from its spleen, which are then fused with myeloma cells (See,
Kohler and Milstein (1976) Eur. J. Immunol. 6: 511-519). The result
is a hybrid cell or "hybridoma" that is capable of reproducing in
vitro.
[0060] Colonies arising from single immortalized cells are screened
for production of antibodies of the desired specificity and
affinity for the antigen, and yield of the monoclonal antibodies
produced by such cells is enhanced by various techniques, including
injection into the peritoneal cavity of a vertebrate host.
Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according to the general protocol
outlined by Huse et al. (1989) Science 246: 1275-1281. In this
manner, the individual antibody species obtained are the products
of immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0061] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, Huse et al. Science
246: 1275-1281 (1989); and Ward, et al. Nature 341: 544-546 (1989).
In general suitable monoclonal antibodies will usually bind their
target epitope with at least a K.sub.D of about 1 mM, more usually
at least about 300 .mu.M, and most preferably at least about 0.1
.mu.M or better.
[0062] C) Circularly Permuted IL-13.
[0063] In another embodiment, the targeting moiety can be a
circularly permuted IL-13 (cpIL-13). Circular permutation is
functionally equivalent to taking a straight-chain molecule, fusing
the ends (directly or through a linker) to form a circular
molecule, and then cutting the circular molecule at a different
location to form a new straight chain molecule with different
termini (see, e.g., Goldenberg, et al. J. Mol. Biol., 165: 407-413
(1983) and Pan et al. Gene 125: 111-114 (1993)). Circular
permutation thus has the effect of essentially preserving the
sequence and identity of the amino acids of a protein while
generating new termini at different locations.
[0064] Circular permutation of IL-13 provides a means by which the
native IL-13 protein may be altered to produce new carboxyl and
amino termini without diminishing the specificity and binding
affinity of the altered first protein relative to its native form.
With new termini located away from the active (binding) site, it is
possible to incorporate the circularly permuted IL-13 into a fusion
protein with a reduced, or no diminution, of IL-13 binding
specificity and/or avidity.
[0065] It will be appreciated that while circular permutation is
described in terms of linking the two ends of a protein and then
cutting the circularized protein these steps are not actually
required to create the end product. A protein can be synthesized de
novo with the sequence corresponding to a circular permutation of
the native protein. Thus, the term "circularly permuted IL-13
(cpIL-13)" refers to all IL-13 proteins having a sequence
corresponding to a circular permutation of a native IL-13 protein
regardless of how they are constructed.
[0066] Generally, however, a permutation that retains or improves
the binding specificity and/or avidity (as compared to the native
IL-13) is preferred. If the new termini interrupt a critical region
of the native protein, binding specificity and avidity may be lost.
Similarly, if linking the original termini destroys IL-13 binding
specificity and avidity then no circular permutation is suitable.
Thus, there are two requirements for the creation of an active
circularly permuted protein: 1) The termini in the native protein
must be favorably located so that creation of a linkage does not
destroy binding specificity and/or avidity; and 2) There must exist
an "opening site" where new termini can be formed without
disrupting a region critical for protein folding and desired
binding activity (see, e.g., Thorton et al. J. Mol. Biol., 167:
443-460 (1983)). This invention establishes that IL-13 meets these
criteria and provides for circularly permuted IL-13 that having
improved binding characteristics.
[0067] When circularly permuting IL-13, it is desirable to use a
linker that preserves the spacing between the termini comparable to
the unpermuted or native molecule. Generally linkers are either
hetero- or homo-bifunctional molecules that contain two reactive
sites that may each form a covalent bond with the carboxyl and the
amino terminal amino acids respectively. Suitable linkers are well
known to those of skill in the art and include, but are not limited
to, straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. The most common and simple example is
a peptide linker that typically consists of several amino acids
joined through peptide bonds to the termini of the native protein.
The linkers may be joined to the terminal amino acids through their
side groups (e.g., through a disulfide linkage to cysteine).
However, in a preferred embodiment, the linkers will be joined to
the alpha carbon amino and carboxyl groups of the terminal amino
acids.
[0068] Functional groups capable of forming covalent bonds with the
amino and carboxyl terminal amino acids are well known to those of
skill in the art. For example, functional groups capable of binding
the terminal amino group include anhydrides, carbodimides, acid
chlorides, activated esters and the like. Similarly, functional
groups capable of forming covalent linkages with the terminal
carboxyl include amines, alcohols, and the like. In a preferred
embodiment, the linker will itself be a peptide and will be joined
to the protein termini by peptide bonds. A preferred linker for the
circular permutation of IL-13 is Gly-Gly-Ser-Gly.
[0069] In a preferred embodiment, circular permutation of IL-13
involves creating an opening such that the formation of new termini
does not interrupt secondary structure crucial to the formation of
a structure that specifically binds the IL-13 receptor. Even if the
three-dimensional structure is compatible with joining the termini,
it is conceivable that the kinetics and thermodynamics of folding
would be greatly altered by circular permutation if the cleavage
separates residues that participate in short range interactions
that are crucial for the folding mechanism or the stability of the
native state. Goldenberg, Protein Eng., 7: 493-495 (1989). Thus,
the choice of a cleavage site can be important to the protein's
binding specificity and/or avidity.
[0070] The selection of an opening site in IL-13 may be determined
by a number of factors. Preferred opening sites will be located in
regions that do not show a highly regular three-dimensional
structure. Thus, it is preferred that cleavage sites be selected in
regions of the protein that do not show secondary structure such as
alpha helices, pleated sheets, .alpha..beta. barrel structures, and
the like.
[0071] Methods of identifying regions of particular secondary
structure of IL-13 based on amino acid sequence are widely known to
those of skill in the art. See, for example, Cohen et al., Science,
263: 488-489 (1994). Numerous programs exist that predict protein
folding based on sequence data. Some of the more widely known
software packages include MatchMaker (Tripos Associates, St. Louis,
Mo., USA), FASMAN from GCG (Genetics Computer Group), PHD (European
Molecular Biology Laboratory, Heidelburg, Germany) and the like. In
addition, the amino acid sequence of IL-13 is well known and the
protein has been extensively characterized (see, e.g., WO
94/04680).
[0072] Alternatively, where the substitution of certain amino acids
or the modification of the side chains of certain amino acids does
not change the activity of a protein, it is expected that the
modified amino acids are not critical to the protein's activity.
Thus, amino acids that are either known to be susceptible to
modification or are actually modified in vivo are potentially good
candidates for cleavage sites.
[0073] Where the protein is a member of a family of related
proteins, one may infer that the highly conserved sequences are
critical for biological activity, while the variable regions are
not. Preferred cleavage sites are then selected in regions of the
protein that do not show highly conserved sequence identity between
various members of the protein family. Alternatively, if a cleavage
site is identified in a conserved region of a protein, that same
region provides a good candidate for cleavage sites in a homologous
protein.
[0074] Methods of determining sequence identity are well known to
those of skill in the art. Sequence comparisons between two (or
more) polynucleotides or polypeptides are typically performed by
comparing sequences of the two sequences over a "comparison window"
to identify and compare local regions of sequence similarity. Since
the goal is to identify very local sequence regions that are not
conserved, the comparison window will be selected to be rather
small. A "comparison window", as used herein, refers to a segment
of at least about 5 contiguous positions, usually about 10 to about
50, more usually about 15 to about 40 in which a sequence may be
compared to a reference sequence of the same number of contiguous
positions after the two sequences are optimally aligned.
[0075] Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith et al. Adv.
Appl. Math. 2: 482 (1981), by the homology alignment algorithm of
Needleman et al., J. Mol. Biol. 48:443 (1970), by the search for
similarity method of Pearson et al., Proc. Natl. Acad. Sci. USA,
85: 2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575
Science Dr., Madison, Wis.), or by inspection.
[0076] A preferred opening site in IL-13 is just prior to Met-44 of
hIL-13, just at the beginning of the putative second alpha-helix
resulting in a circularly permuted IL-13 having a methionine at
position 44 of the native IL-13 at the amino terminus of the
cpIL-13 and the Glycine at position 43 of the native IL-13 at the
new carboxyl terminus of the cpIL-13. This carboxyl terminus can be
joined to a second protein directly or though a spacer.
[0077] Circularly permuted IL-13 may be made by a number of means
known to those of skill in the art. These include chemical
synthesis, modification of existing proteins, and expression of
circularly permuted proteins using recombinant DNA methodology.
[0078] The circularly permuted IL-13 may be synthesized using
standard chemical peptide synthesis techniques as discussed below
in section IV(B). If the linker is a peptide it may be incorporated
during the synthesis. If the linker is not a peptide it may be
coupled to the peptide after synthesis.
[0079] Alternatively, the circularly permuted IL-13 can be made by
chemically modifying a native IL-13 (e.g. a native human IL-13).
Generally, this requires reacting the IL-13 in the presence of the
linker to form covalent bonds between the linker and the carboxyl
and amino termini of the protein, thus forming a circular protein.
New termini are then formed by cleaving the peptide bond joining
amino acids at another location. This may be accomplished
chemically or enzymatically using, for example, a peptidase.
[0080] If the cleavage reaction tends to hydrolyze more than one
peptide bond, the reaction may be run briefly. Those molecules
having more than one peptide bond cleaved will be shorter than the
full length circularly permuted molecule and the latter may be
isolated by any protein purification technique that selects by size
(e.g., by size exclusion chromatography or electrophoresis).
Alternatively, various sites in the circular protein may be
protected from hydrolysis by chemical modification of the amino
acid side chains which may interfere with enzyme binding, or by
chemical blocking of the vulnerable groups participating in the
peptide bond.
[0081] In a preferred embodiment, the circularly permuted IL-13, or
fusion proteins comprising the circularly permuted IL-13 will be
synthesized using recombinant DNA methodology. Generally this
involves creating a DNA sequence that encodes the circularly
permuted growth factor (or entire fusion protein containing the
growth factor), placing the DNA in an expression cassette under the
control of a particular promoter, expressing the protein in a host,
isolating the expressed protein and, if required, renaturing the
protein. Recombinant expression of the fusion proteins of this
invention is discussed in more detail below in section IV(B).
[0082] DNA encoding circularly permuted growth factors or fusion
proteins comprising circularly permuted growth factors may be
prepared by any suitable method, including, for example, cloning
and restriction of appropriate sequences or direct chemical
synthesis by methods as discussed below. Alternatively,
subsequences may be cloned and the appropriate subsequences cleaved
using appropriate restriction enzymes. The fragments may then be
ligated to produce the desired DNA sequence.
[0083] In a preferred embodiment, DNA encoding the circularly
permuted growth factor may be produced using DNA amplification
methods, for example polymerase chain reaction (PCR). First, the
segments of the native DNA on either side of the new terminus are
amplified separately. The 5' end of the one amplified sequence
encodes the peptide linker, while the 3' end of the other amplified
sequence also encodes the peptide linker. Since the 5' end of the
first fragment is complementary to the 3' end of the second
fragment, the two fragments (after partial purification, e.g. on
LMP agarose) can be used as an overlapping template in a third PCR
reaction. The amplified sequence will contain codons the segment on
the carboxy side of the opening site (now forming the amino
sequence), the linker, and the sequence on the amino side of the
opening site (now forming the carboxyl sequence). The circularly
permuted molecule may then be ligated into a plasmid and expressed
as discussed below.
[0084] D) Modified IL-13.
[0085] One of skill in the art will appreciate that IL-13 can be
modified in a variety of ways that do not destroy binding
specificity and/or avidity and, in fact, may increase binding
properties. Some modifications may be made to facilitate the
cloning, expression, or incorporation of the circularly permuted
growth factor into a fusion protein. Such modifications are well
known to those of skill in the art and include, for example, a
methionine added at the amino terminus to provide an initiation
site, or additional amino acids placed on either terminus to create
conveniently located restriction sites or termination codons.
[0086] One of skill will recognize that other modifications may be
made. Thus, for example, amino acid substitutions may be made that
increase specificity or binding affinity of the circularly permuted
protein, etc. Alternatively, non-essential regions of the molecule
may be shortened or eliminated entirely. Thus, where there are
regions of the molecule that are not themselves involved in the
activity of the molecule, they may be eliminated or replaced with
shorter segments that merely serve to maintain the correct spatial
relationships between the active components of the molecule.
[0087] E) Other Targeting Antibodies.
[0088] Where the chimeric molecule contains more than one targeting
molecule (e.g. a dual-targeted cytotoxin), the molecule may contain
targeting antibodies directed to tumor markers other than the
overexpressed IL-13 receptor. A number of such antibodies are known
and have even been converted to form suitable for incorporation
into fusion proteins. These include anti-erbB2, B3, BR96, OVB3,
anti-transferrin, Mik-.beta.1 and PR1 (see Batra et al., Mol. Cell
Biol., 11: 2200-2205 (1991); Batra et al., Proc. Natl. Acad. Sci.
USA, 89: 5867-5871 (1992); Brinkmann, et al. Proc. Natl. Acad. Sci.
USA, 88: 8616-8620 (1991); Brinkmann et al., Proc. Natl. Acad. Sci.
USA, 90: 547-551 (1993); Chaudhary et al., Proc. Natl. Acad. Sci.
USA, 87: 1066-1070 (1990); Friedman et al., Cancer Res. 53: 334-339
(1993); Kreitman et al., J. Immunol., 149: 2810-2815 (1992);
Nicholls et al., J. Biol. Chem., 268: 5302-5308 (1993); and Wells,
et al., Cancer Res., 52: 6310-6317 (1992), respectively).
[0089] III. The Effector Molecule.
[0090] As described above, the effector molecule component of the
chimeric molecules of this invention may be any molecule whose
activity it is desired to deliver to cells that overexpress IL-13
receptors. Particularly preferred effector molecules include
cytotoxins such as PE or DT, radionuclides, ligands such as growth
factors, antibodies, detectable labels such as fluorescent or
radioactive labels, and therapeutic compositions such as liposomes
and various drugs.
[0091] A) Cytotoxins.
[0092] Particularly preferred cytotoxins include Pseudomonas
exotoxins, Diphtheria toxins, ricin, and abrin. Pseudomonas
exotoxin and Dipthteria toxin are most preferred.
[0093] i) Pseudomonas Exotoxin (PE).
[0094] Pseudomonas exotoxin A (PE) is an extremely active monomeric
protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells
through the inactivation of elongation factor 2 (EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP
ribosyl moiety of oxidized NAD onto EF-2).
[0095] The toxin contains three structural domains that act in
concert to cause cytotoxicity. Domain Ia (amino acids 1-252)
mediates cell binding. Domain II (amino acids 253-364) is
responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2, which inactivates the protein and causes cell death. The
function of domain lb (amino acids 365-399) remains undefined,
although a large part of it, amino acids 365-380, can be deleted
without loss of cytotoxicity. See Siegall et al., J. Biol. Chem.
264: 14256-14261 (1989).
[0096] Where the targeting molecule (e.g. IL-13) is fused to PE, a
preferred PE molecule is one in which domain Ia (amino acids 1
through 252) is deleted and amino acids 365 to 380 have been
deleted from domain lb. However all of domain lb and a portion of
domain II (amino acids 350 to 394) can be deleted, particularly if
the deleted sequences are replaced with a linking peptide such as
GGGGS.
[0097] In addition, the PE molecules can be further modified using
site-directed mutagenesis or other techniques known in the art, to
alter the molecule for a particular desired application. Means to
alter the PE molecule in a manner that does not substantially
affect the functional advantages provided by the PE molecules
described here can also be used and such resulting molecules are
intended to be covered herein.
[0098] For maximum cytotoxic properties of a preferred PE molecule,
several modifications to the molecule are recommended. An
appropriate carboxyl terminal sequence to the recombinant molecule
is preferred to translocate the molecule into the cytosol of target
cells. Amino acid sequences which have been found to be effective
include, REDLK (as in native PE), REDL, RDEL, or KDEL, repeats of
those, or other sequences that function to maintain or recycle
proteins into the endoplasmic reticulum, referred to here as
"endoplasmic retention sequences". See, for example, Chaudhary et
al, Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam et al, J.
Biol. Chem. 266: 17376-17381 (1991).
[0099] Deletions of amino acids 365-380 of domain lb can be made
without loss of activity. Further, a substitution of methionine at
amino acid position 280 in place of glycine to allow the synthesis
of the protein to begin and of serine at amino acid position 287 in
place of cysteine to prevent formation of improper disulfide bonds
is beneficial.
[0100] In a preferred embodiment, the targeting molecule is
inserted in replacement for domain Ia. A similar insertion has been
accomplished in what is known as the TGF.alpha.-PE40 molecule (also
referred to as TP40) described in Heimbrook et al., Proc. Natl.
Acad. Sci., USA, 87: 4697-4701 (1990) and in U.S. Pat. No.
5,458,878.
[0101] Those skilled in the art will realize that additional
modifications, deletions, insertions and the like may be made to
the chimeric molecules of the present invention or to the nucleic
acid sequences encoding IL-13 receptor-directed chimeric molecules.
Especially, deletions or changes may be made in PE or in a linker
connecting an antibody gene to PE, in order to increase
cytotoxicity of the fusion protein toward target cells or to
decrease nonspecific cytotoxicity toward cells without antigen for
the antibody. All such constructions may be made by methods of
genetic engineering well known to those skilled in the art (see,
generally, Sambrook et al., supra) and may produce proteins that
have differing properties of affinity, specificity, stability and
toxicity that make them particularly suitable for various clinical
or biological applications.
[0102] ii) Diphtheria Toxin (DT).
[0103] Like PE, diphtheria toxin (DT) kills cells by
ADP-ribosylating elongation factor 2 thereby inhibiting protein
synthesis. Diphtheria toxin, however, is divided into two chains, A
and B, linked by a disulfide bridge. In contrast to PE, chain B of
DT, which is on the carboxyl end, is responsible for receptor
binding and chain A, which is present on the amino end, contains
the enzymatic activity (Uchida et al., Science, 175: 901-903
(1972); Uchida et al. J. Biol. Chem., 248: 3838-3844 (1973)).
[0104] In a preferred embodiment, the targeting molecule-Diphtheria
toxin fusion proteins of this invention have the native
receptor-binding domain removed by truncation of the Diphtheria
toxin B chain. Particularly preferred is DT388, a DT in which the
carboxyl terminal sequence beginning at residue 389 is removed.
Chaudhary, et al., Bioch. Biophys. Res. Comm., 180: 545-551
(1991).
[0105] Like the PE chimeric cytotoxins, the DT molecules may be
chemically conjugated to the IL-13 receptor targeting molecule,
but, in a preferred embodiment, the targeting molecule will be
fused to the Diphtheria toxin by recombinant means. The genes
encoding protein chains may be cloned in cDNA or in genomic form by
any cloning procedure known to those skilled in the art. Methods of
cloning genes encoding DT fused to various ligands are also well
known to those of skill in the art (see, e.g., Williams et al. J.
Biol. Chem. 265: 11885-11889 (1990)).
[0106] The term "Diphtheria toxin" (DT) as used herein refers to
full length native DT or to a DT that has been modified.
Modifications typically include removal of the targeting domain in
the B chain and, more specifically, involve truncations of the
carboxyl region of the B chain.
[0107] B) Detectable Labels.
[0108] Detectable labels suitable for use as the effector molecule
component of the chimeric molecules of this invention include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include magnetic beads (e.g.
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
texas red, rhodamine, green fluorescent protein, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0109] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0110] C) Ligands.
[0111] As explained above, the effector molecule may also be a
ligand or an antibody. Particularly preferred ligand and antibodies
are those that bind to surface markers on immune cells. Chimeric
molecules utilizing such antibodies as effector molecules act as
bifunctional linkers establishing an association between the immune
cells bearing binding partner for the ligand or antibody and the
tumor cells overexpressing the IL-13 receptor. Suitable antibodies
and growth factors are known to those of skill in the art and
include, but are not limited to, IL-2, 1L-4, IL-6, IL-7, tumor
necrosis factor (TNF), anti-Tac, TGF.alpha., and the like.
[0112] D) Other Therapeutic Moieties.
[0113] Other suitable effector molecules include pharmacological
agents or encapsulation systems containing various pharmacological
agents. Thus, the targeting molecule of the chimeric molecule may
be attached directly to a drug that is to be delivered directly to
the tumor. Such drugs are well known to those of skill in the art
and include, but are not limited to, doxirubicin, vinblastine,
genistein, an antisense molecule, and the like.
[0114] Alternatively, the effector molecule may be an encapsulation
system, such as a liposome or micelle that contains a therapeutic
composition such as a drug, a nucleic acid (e.g. an antisense
nucleic acid), or another therapeutic moiety that is preferably
shielded from direct exposure to the circulatory system. Means of
preparing liposomes attached to antibodies are well known to those
of skill in the art. See, for example, U.S. Pat. No. 4,957,735,
Connor et al., Pharm. Ther., 28: 341-365 (1985)
[0115] IV. Attachment of the Targeting Molecule to the Effector
Molecule.
[0116] One of skill will appreciate that the targeting molecule and
effector molecules may be joined together in any order. Thus, where
the targeting molecule is a polypeptide, the effector molecule may
be joined to either the amino or carboxy termini of the targeting
molecule. The targeting molecule may also be joined to an internal
region of the effector molecule, or conversely, the effector
molecule may be joined to an internal location of the targeting
molecule, as long as the attachment does not interfere with the
respective activities of the molecules.
[0117] The targeting molecule and the effector molecule may be
attached by any of a number of means well known to those of skill
in the art. Typically the effector molecule is conjugated, either
directly or through a linker (spacer), to the targeting molecule.
However, where both the effector molecule and the targeting
molecule are polypeptides it is preferable to recombinantly express
the chimeric molecule as a single-chain fusion protein.
[0118] A) Conjugation of the Effector Molecule to the Targeting
Molecule.
[0119] Preferred forms of PE contain amino acids 253-364 and
381-608, and are followed by the native sequences REDLK or the
mutant sequences KDEL or RDEL. Lysines at positions 590 and 606 may
or may not be mutated to glutamine.
[0120] In a particularly preferred embodiment, the IL-13 receptor
targeted cytotoxins of this invention comprise the PE molecule
designated PE38QQR. This PE molecule is a truncated form of PE
composed of amino acids 253-364 and 381-608. The lysine residues at
positions 509 and 606 are replaced by glutamine and at 613 are
replaced by arginine (Debinski et al. Bioconj. Chem., 5: 40
(1994)).
[0121] In another particularly preferred embodiment, the IL-13
receptor targeted cytotoxins of this invention comprise the PE
molecule designated PE4E. PE4E is a "full length" PE with a mutated
and inactive native binding domain where amino acids 57, 246, 247,
and 249 are all replaced by glutamates (see, e.g., Chaudhary et
al., J. Biol. Chem., 265: 16306 (1995)).
[0122] The targeting molecule (e.g. IL-13 or anti-IL-13R antibody)
may also be inserted at a point within domain III of the PE
molecule. Most preferably the targeting molecule is fused between
about amino acid positions 607 and 609 of the PE molecule. This
means that the targeting molecule is inserted after about amino
acid 607 of the molecule and an appropriate carboxyl end of PE is
recreated by placing amino acids about 604-613 of PE after the
targeting molecule. Thus, the targeting molecule is inserted within
the recombinant PE molecule after about amino acid 607 and is
followed by amino acids 604-613 of domain III. The targeting
molecule may also be inserted into domain Ib to replace sequences
not necessary for toxicity. Debinski, et al. Mol. Cell. Biol., 11:
1751-1753 (1991).
[0123] In a preferred embodiment, the PE molecules will be fused to
the targeting molecule by recombinant means. The genes encoding
protein chains may be cloned in cDNA or in genomic form by any
cloning procedure known to those skilled in the art (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, (1989)). Methods of cloning genes
encoding PE fused to various ligands are well known to those of
skill in the art (see, e.g., Siegall et al., FASEB J., 3: 2647-2652
(1989); and Chaudhary et al. Proc. Natl. Acad. Sci. USA, 84:
4538-4542 (1987)).
[0124] In one embodiment, the targeting molecule (e.g., IL-13,
cpIL-13, or anti-IL-13R antibody) is chemically conjugated to the
effector molecule (e.g., a cytotoxin, a label, a ligand, or a drug
or liposome). Means of chemically conjugating molecules are well
known to those of skill.
[0125] The procedure for attaching an agent to an antibody or other
polypeptide targeting molecule will vary according to the chemical
structure of the agent. Polypeptides typically contain variety of
functional groups; e.g., carboxylic acid (COOH) or free amine
(--NH.sub.2) groups, which are available for reaction with a
suitable functional group on an effector molecule to bind the
effector thereto.
[0126] Alternatively, the targeting molecule and/or effector
molecule may be derivatized to expose or attach additional reactive
functional groups. The derivatization may involve attachment of any
of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford Ill.
[0127] A "linker", as used herein, is a molecule that is used to
join the targeting molecule to the effector molecule. The linker is
capable of forming covalent bonds to both the targeting molecule
and to the effector molecule. Suitable linkers are well known to
those of skill in the art and include, but are not limited to,
straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Where the targeting molecule and the
effector molecule are polypeptides, the linkers may be joined to
the constituent amino acids through their side groups (e.g.,
through a disulfide linkage to cysteine). However, in a preferred
embodiment, the linkers will be joined to the alpha carbon amino
and carboxyl groups of the terminal amino acids.
[0128] A bifunctional linker having one functional group reactive
with a group on a particular agent, and another group reactive with
an antibody, may be used to form the desired immunoconjugate.
Alternatively, derivatization may involve chemical treatment of the
targeting molecule, e.g., glycol cleavage of the sugar moiety of a
the glycoprotein antibody with periodate to generate free aldehyde
groups. The free aldehyde groups on the antibody may be reacted
with free amine or hydrazine groups on an agent to bind the agent
thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation
of free sulfhydryl groups on polypeptide, such as antibodies or
antibody fragments, are also known (See U.S. Pat. No.
4,659,839).
[0129] Many procedure and linker molecules for attachment of
various compounds including radionuclide metal chelates, toxins and
drugs to proteins such as antibodies are known. See, for example,
European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and
4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987).
In particular, production of various immunotoxins is well-known
within the art and can be found, for example in "Monoclonal
Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al.,
Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.
168-190 (1982), Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos.
4,545,985 and 4,894,443.
[0130] In some circumstances, it is desirable to free the effector
molecule from the targeting molecule when the chimeric molecule has
reached its target site. Therefore, chimeric conjugates comprising
linkages which are cleavable in the vicinity of the target site may
be used when the effector is to be released at the target site.
Cleaving of the linkage to release the agent from the antibody may
be prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in
the vicinity of the target site. When the target site is a tumor, a
linker which is cleavable under conditions present at the tumor
site (e.g. when exposed to tumor-associated enzymes or acidic pH)
may be used.
[0131] A number of different cleavable linkers are known to those
of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and
4,625,014. The mechanisms for release of an agent from these linker
groups include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example,
includes a description of immunoconjugates comprising linkers which
are cleaved at the target site ill vivo by the proteolytic enzymes
of the patient's complement system. In view of the large number of
methods that have been reported for attaching a variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs,
toxins, and other agents to antibodies one skilled in the art will
be able to determine a suitable method for attaching a given agent
to an antibody or other polypeptide.
[0132] B) Production of Fusion Proteins.
[0133] Where the targeting molecule and/or the effector molecule is
relatively short (i.e., less than about 50 amino acids) they may be
synthesized using standard chemical peptide synthesis techniques.
Where both molecules are relatively short the chimeric molecule may
be synthesized as a single contiguous polypeptide. Alternatively
the targeting molecule and the effector molecule may be synthesized
separately and then fused by condensation of the amino terminus of
one molecule with the carboxyl terminus of the other molecule
thereby forming a peptide bond. Alternatively, the targeting and
effector molecules may each be condensed with one end of a peptide
spacer molecule thereby forming a contiguous fusion protein.
[0134] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2:
Special Methods in Peptide Synthesis, Part A., Merrifield, et al.
J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid
Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
(1984).
[0135] In a preferred embodiment, the chimeric fusion proteins of
the present invention are synthesized using recombinant DNA
methodology. Generally this involves creating a DNA sequence that
encodes the fusion protein, placing the DNA in an expression
cassette under the control of a particular promoter, expressing the
protein in a host, isolating the expressed protein and, if
required, renaturing the protein.
[0136] DNA encoding the fusion proteins (e.g. IL-13-PE38QQR) of
this invention may be prepared by any suitable method, including,
for example, cloning and restriction of appropriate sequences or
direct chemical synthesis by methods such as the phosphotriester
method of Narang et al. Meth. Enzymol. 68: 90-99 (1979); the
phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151
(1979); the diethylphosphoramidite method of Beaucage et al.,
Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of
U.S. Pat. No. 4,458,066.
[0137] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0138] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0139] In a preferred embodiment, DNA encoding fusion proteins of
the present invention may be cloned using DNA amplification methods
such as polymerase chain reaction (PCR). Thus, in a preferred
embodiment, the gene for IL-13 is PCR amplified, using a sense
primer containing the restriction site for NdeI and an antisense
primer containing the restriction site for HindIII. In a
particularly preferred embodiment, the primers are selected to
amplify the nucleic acid starting at position 19, as described by
McKenzie et al. (1987), supra. This produces a nucleic acid
encoding the mature IL-13 sequence and having terminal restriction
sites. A PE38QQR fragment may be cut out of the plasmid
pWDMH4-38QQR or plasmid pSGC242FdN1 described by Debinski et al.
Int. J. Cancer, 58: 744-748 (1994), and by Debinski et al. Clin.
Res., 42: 251A (abstract (1994) respectively. Ligation of the IL-13
and PE38QQR sequences and insertion into a vector produces a vector
encoding IL-13 joined to the amino terminus of PE38QQR (position
253 of PE). The two molecules are joined by a three amino acid
junction consisting of glutamic acid, alanine, and phenylalanine
introduced by the restriction site.
[0140] While the two molecules are preferably essentially directly
joined together, one of skill will appreciate that the molecules
may be separated by a peptide spacer consisting of one or more
amino acids. Generally the spacer will have no specific biological
activity other than to join the proteins or to preserve some
minimum distance or other spatial relationship between them.
However, the constituent amino acids of the spacer may be selected
to influence some property of the molecule such as the folding, net
charge, or hydrophobicity.
[0141] The nucleic acid sequences encoding the fusion proteins may
be expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and various higher eukaryotic cells such as
the COS, CHO and HeLa cells lines and myeloma cell lines. The
recombinant protein gene will be operably linked to appropriate
expression control sequences for each host. For E. coli this
includes a promoter such as the 17, trp, or lambda promoters, a
ribosome binding site and preferably a transcription termination
signal. For eukaryotic cells, the control sequences will include a
promoter and preferably an enhancer derived from immunoglobulin
genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence,
and may include splice donor and acceptor sequences.
[0142] The plasmids of the invention can be transferred into the
chosen host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0143] Once expressed, the recombinant fusion proteins can be
purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like (see, generally,
R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982),
Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification., Academic Press, Inc. N.Y. (1990)). Substantially
pure compositions of at least about 90 to 95% homogeneity are
preferred, and 98 to 99% or more homogeneity are most preferred for
pharmaceutical uses. Once purified, partially or to homogeneity as
desired, the polypeptides may then be used therapeutically.
[0144] One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the IL-13
receptor targeted fusion protein may possess a conformation
substantially different than the native conformations of the
constituent polypeptides. In this case, it may be necessary to
denature and reduce the polypeptide and then to cause the
polypeptide to re-fold into the preferred conformation. Methods of
reducing and denaturing proteins and inducing re-folding are well
known to those of skill in the art (See, Debinski et al. J. Biol.
Chem., 268: 14065-14070 (1993); Kreitman and Pastan, Bioconjug.
Chem., 4: 581-585 (1993); and Buchner, et al., Anal. Biochem., 205:
263-270 (1992)). Debinski et al., for example, describe the
denaturation and reduction of inclusion body proteins in
guanidine-DTE. The protein is then refolded in a redox buffer
containing oxidized glutathione and L-arginine.
[0145] One of skill would recognize that modifications can be made
to the IL-13 receptor targeted fusion proteins without diminishing
their biological activity. Some modifications may be made to
facilitate the cloning, expression, or incorporation of the
targeting molecule into a fusion protein. Such modifications are
well known to those of skill in the art and include, for example, a
methionine added at the amino terminus to provide an initiation
site, or additional amino acids placed on either terminus to create
conveniently located restriction sites or termination codons.
[0146] V. Identification Of Target Cells.
[0147] It was a surprising discovery of the present invention that
tumor cells, overexpress IL-13 receptors. In particular, carcinoma
tumor cells (e.g. renal carcinoma cells) overexpress IL-13
receptors at levels ranging from about 2100 sites/cell to greater
than 150,000 sites per cell. Similarly, gliomas and Kaposi's
sarcoma also overexpress IL-13 receptors (L-13R). Moreover, every
cancer type tested to date appears to overexpress IL-13 receptors.
Thus it appears that IL-13 receptor overexpression is general
characteristic of a solid tumor neoplastic cell.
[0148] Thus, the methods of this invention can be used to target an
effector molecule to virtually any neoplastic cell. Neoplasias are
well known to those of skill in the art and include, but are not
limited to, cancers of the skin (e.g., basal or squamous cell
carcinoma, melanoma, Kaposi's sarcoma, etc.), cancers of the
reproductive system (e.g., testicular, ovarian, cervical), cancers
of the gastrointestinal tract (e.g., stomach, small intestine,
large intestine, colorectal, etc.), cancers of the mouth and throat
(e.g. esophageal, larynx, oropharynx, nasopharynx, oral, etc.),
cancers of the head and neck, bone cancers, breast cancers, liver
cancers, prostate cancers (e.g., prostate carcinoma), thyroid
cancers, heart cancers, retinal cancers (e.g., melanoma), kidney
cancers, lung cancers (e.g., mesothelioma), pancreatic cancers,
brain cancers (e.g. gliomas, medulloblastomas, pituitary ademomas,
etc.) and cancers of the lymph system (e.g. lymphoma).
[0149] In a particularly preferred embodiment, the methods of this
invention are used to target effector molecules to kidney cancers,
colorectal cancers (especially colorectal carcinomas), to skin
cancers (especially Kaposi's sarcoma), and to brain cancers
(especially gliomas, and medulloblastomas).
[0150] One of skill in the art will appreciate that identification
and confirmation of IL-13 overexpression by other cells requires
only routine screening using well-known methods. Typically this
involves providing a labeled molecule that specifically binds to
the IL-13 receptor. The cells in question are then contacted with
this molecule and washed. Quantification of the amount of label
remaining associated with the test cell provides a measure of the
amount of IL-13 receptor (IL-13R) present on the surface of that
cell.
[0151] In a preferred embodiment, IL-13 receptor may be quantified
by measuring the binding of .sup.125I-labeled IL-13
(.sup.125I-IL-13) to the cell in question. Details of such a
binding assay are provided in Example 1.
[0152] VI. Pharmaceutical Compositions.
[0153] The chimeric molecules of this invention are useful for
parenteral, topical, oral, or local administration, such as by
aerosol or transdermally, for prophylactic and/or therapeutic
treatment. The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include powder, tablets, pills, capsules and
lozenges. It is recognized that the fusion proteins and
pharmaceutical compositions of this invention, when administered
orally, must be protected from digestion. This is typically
accomplished either by complexing the protein with a composition to
render it resistant to acidic and enzymatic hydrolysis or by
packaging the protein in an appropriately resistant carrier such as
a liposome. Means of protecting proteins from digestion are well
known in the art.
[0154] The pharmaceutical compositions of this invention are
particularly useful for parenteral administration, such as
intravenous administration or administration into a body cavity or
lumen of an organ. The compositions for administration will
commonly comprise a solution of the chimeric molecule dissolved in
a pharmaceutically acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., buffered
saline and the like. These solutions are sterile and generally free
of undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of chimeric molecule in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0155] Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
[0156] The compositions containing the present fusion proteins or a
cocktail thereof (i.e., with other proteins) can be administered
for therapeutic treatments. In therapeutic applications,
compositions are administered to a patient suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. An amount adequate to
accomplish this is defined as a "therapeutically effective dose."
Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health.
[0157] Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
[0158] Among various uses of the cytotoxic fusion proteins of the
present invention are included a variety of disease conditions
caused by specific human cells that may be eliminated by the toxic
action of the protein. One preferred application is the treatment
of cancer, such as by the use of an IL-13 receptor targeting
molecule (e.g. IL-13 or anti-IL-13R antibody) attached to a
cytotoxin.
[0159] Where the chimeric molecule comprises an IL-13 receptor
targeting molecule attached to a ligand, ligand portion of the
molecule is chosen according to the intended use. Proteins on the
membranes of T cells that may serve as targets for the ligand
includes CD2 (T11), CD3, CD4 and CD8. Proteins found predominantly
on B cells that might serve as targets include CD10 (CALLA
antigen), CD19 and CD20. CD45 is a possible target that occurs
broadly on lymphoid cells. These and other possible target
lymphocyte target molecules for the chimeric molecules bearing a
ligand effector are described in Leukocyte Typing III, A. J.
McMichael, ed., Oxford University Press (1987). Those skilled in
the art will realize ligand effectors may be chosen that bind to
receptors expressed on still other types of cells as described
above, for example, membrane glycoproteins or ligand or hormone
receptors such as epidermal growth factor receptor and the
like.
[0160] It will be appreciated by one of skill in the art that there
are some regions that are not heavily vascularized or that are
protected by cells joined by tight junctions and/or active
transport mechanisms which reduce or prevent the entry of
macromolecules present in the blood stream. Thus, for example,
systemic administration of therapeutics to treat gliomas, or other
brain cancers, is constrained by the blood-brain barrier which
resists the entry of macromolecules into the subarachnoid
space.
[0161] One of skill in the art will appreciate that in these
instances, the therapeutic compositions of this invention can be
administered directly to the tumor site. Thus, for example, brain
tumors (e.g., gliomas) can be treated by administering the
therapeutic composition directly to the tumor site (e.g., through a
surgically implanted catheter). Where the fluid delivery through
the catheter is pressurized, small molecules (e.g. the therapeutic
molecules of this invention) will typically infiltrate as much as
two to three centimeters beyond the tumor margin.
[0162] Alternatively, the therapeutic composition can be placed at
the target site in a slow release formulation. Such formulations
can include, for example, a biocompatible sponge or other inert or
resorbable matrix material impregnated with the therapeutic
composition, slow dissolving time release capsules or
microcapsules, and the like.
[0163] Typically the catheter or time release formulation will be
placed at the tumor site as part of a surgical procedure. Thus, for
example, where major tumor mass is surgically removed, the
perfusing catheter or time release formulation can be emplaced at
the tumor site as an adjunct therapy. Of course, surgical removal
of the tumor mass may be undesired, not required, or impossible, in
which case, the delivery of the therapeutic compositions of this
invention may comprise the primary therapeutic modality.
[0164] VII. Diagnostic Kits.
[0165] In another embodiment, this invention provides for kits for
the treatment of tumors or for the detection of cells
overexpressing IL-13 receptors. Kits will typically comprise a
chimeric molecule of the present invention (e.g. IL-13-label,
IL-13-cytotoxin, IL-13-ligand, etc.). In addition the kits will
typically include instructional materials disclosing means of use
of chimeric molecule (e.g. as a cytotoxin, for detection of tumor
cells, to augment an immune response, etc.). The kits may also
include additional components to facilitate the particular
application for which the kit is designed. Thus, for example, where
a kit contains a chimeric molecule in which the effector molecule
is a detectable label, the kit may additionally contain means of
detecting the label (e.g. enzyme substrates for enzymatic labels,
filter sets to detect fluorescent labels, appropriate secondary
labels such as a sheep anti-mouse-HRP, or the like). The kits may
additionally include buffers and other reagents routinely used for
the practice of a particular method. Such kits and appropriate
contents are well known to those of skill in the art.
EXAMPLES
[0166] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Identification of Cells that Overexpress IL-13
[0167] Recombinant human IL-4 and IL-13 were labeled with .sup.125I
(Amersham Research Products, Arlington Heights, Ill., USA) by using
the IODO-GEN reagent (Pierce, Rockford, Ill., USA) according to the
manufacturer's instructions. The specific activity of the
radiolabeled cytokines was estimated to range from 20-100
.mu.Ci/.mu.g protein. For binding experiments, typically,
1.times.10.sup.6 renal cell carcinoma (RCC) tumor cells were
incubated at 4.degree. C. for 2 hours with .sup.125I-IL-13 (100 pM)
with or without increasing concentrations (up to 500 nM) of
unlabeled IL-13. In some experiments, IL-13R expression was
examined as previously described (Obiri et al. J. Clin. Invest.,
91: 88-93 (1993))). The data were analyzed with the LIGAND program
(Munson et al. Anal. Biochem., 107: 220-239 (1980)) to determine
receptor number and binding affinity.
[0168] Four human renal cell carcinoma (RCC) cell lines (WS-RCC,
HL-RCC, PM-RCC, and MA-RCC) bound .sup.125I-IL-13 specifically and
the density of IL-13R varied from 2100 sites per cell in WS-RCC
cells to 150,000 sites per cell in HL-RCC cells (Table 1). The
represents an increase in IL-13 receptor expression ranging from 15
to about 500 fold as compared to normal immune cells. In contrast,
IL-4 receptors overexpressed on cancers have been reported at
concentrations as high as 4000 sites per cell. Scatchard analyses
(Scatchard, Ann. N.Y. Acad. Sci., 51: 660-663 (1949)) revealed that
only one affinity class of receptors was expressed on each cell
line. The binding affinities (Kd) ranged between 100 pM to 400 pM
in three RCC cell lines while HL-RCC cells expressed lower affinity
receptors (Kd .sup.-3 nM).
[0169] Although IL-13 responsiveness has previously been reported
in human monocytes, B cells and pre-myeloid (TF-1) cells (see, e.g.
de Waal Malefyt, et al. J. Immunol., 151: 6370-6381 (1993), de Waal
Malefyt, et al. J. Immunol., 144: 629-633 (1993)), little was known
about IL-13R structure or its binding characteristics in these, or
any other cells. The present data show that freshly isolated human
monocytes, EBV-transformed B cell line and TF-1 cell line express
very few IL-13 binding sites (100-300/cell) compared to human RCC
cells (Table 1). On the other hand, no binding of .sup.125I-IL-13
was observed on H9 T cells, LAK cells and resting or PHA activated
PBL. This is compatible with the fact that IL-13 responsiveness has
not been observed in T lymphocytes (Punnonen et al., Proc. Natl.
Acad. Sci. USA, 90: 3730-3734 (1993).
1TABLE 1 Expression of IL-13 receptor by human cells. IL-13 Binding
Sites/cell.sup.a Kd (nM) Cell Types Mean .+-. SD Mean .+-. SD Renal
Cell Carcinoma (RCC) 1. WS-RCC 2,090 .+-. 367 (5) 0.247 .+-. 0.12
(3).sup.b 2. MA-RCC 5,013 .+-. 1.347 (5) 0.128 .+-. 0.05 (2) 3.
PM-RCC 26,500 .+-. 5.000 (2) 0.394 .+-. 0.26 (2) 4. HL-RCC 150,000
.+-. 15.00 (3) 3.1 .+-. 0.7 (2) B Lymphocytes 1. DH
(EBV-transformed B 303 .+-. 90 (4) --.sup.d cell line) 2. RAJI
(Burkitt's UD.sup.c -- lymphoma) Monocytes/Premyeloid cells.sup.e
1. Peripheral blood 124 -- monocytes 2. U937 (premonocytic UD -- 3.
TF1.J61 (premyeloid) 130 .+-. 1 (2) -- T Lymphocytes/LAK
cells.sup.f 1. PHA-activated PBL <30 -- 2. MOLT-4 (T-cell UD --
leukemia) 3. LAK cells UD -- .sup.aIL-13 binding sites/cell were
determined as described in Example 1. .sup.b(n) = number of
experiments used to calculate mean .+-. standard deviation.
.sup.cUC = undetectable .sup.dThe Kd could not be reliably
calculated because of low binding of .sup.125I-IL-13 .sup.eThe
peripheral blood derived monocytes (>90% purity) were isolated
by ficoll-hypaque density gradient followed by ellutriation from a
leukopac obtained from normal donor. .sup.fLAK cells and activated
T-lymphocytes were generated by the culture of donor PBLs (106/ml)
with IL-2 (500 Units/ml) for 3 days or PHA (10 .mu.g/ml) for 3-4
days respectively.
Example 2
IL-13 and IL-4 Bind to Different Receptors
[0170] Recently, it was proposed that the IL-2R.gamma..sub.c
receptor subunit is associated with IL-13R (see, e.g., Russell et
al. Science 262: 1880-1883 (1993); Kondo et al. Science, 262:
1874-1877 (1993); Noguchi et al. Science, 262: 1877-1880 (1993);
Kondo et al. Science 263: 1453-1454 (1994); Giri et al. EMBO J. 13:
2822-2830 (1994))) and IL-13R may share a common component with
IL-4R (Zurawski et al. EMBO J. 12: 2663-2670 (1993); Aversa et al.
J. Exp. Med. 178: 2213-2218 (1993)). To directly address these
possibilities, radio-ligand binding experiments were performed, as
described in Example 1, on HL-RCC and WS-RCC cells using
.sup.125I-IL-4 or .sup.125I-IL-13 in the presence or absence of
excess of either cytokine.
[0171] Unlabeled IL-4 more efficiently inhibited .sup.125I-IL-4
from binding to RCC cells (84%, and 72% displacement of total
binding in WS-RCC and HL-RCC, respectively) than IL-13 which also
displaced .sup.125I-IL-4 binding to these cells (61% of total
binding in WS-RCC and 51% in HL-RCC) under similar conditions. On
the other hand, while .sup.125I-IL-13 binding was effectively
displaced by IL-13 (about 85% of total in both cell types), it was
only minimally displaced by IL-4 (12% of total displacement in
WS-RCC, and 7% in HL-RCC). These results indicate that IL-4 and
IL-13 both interact with each other's receptors, however, the
interaction is not identical since IL-4 inhibition of
.sup.125I-IL-13 binding was weak and IL-13 inhibition of
.sup.125I-IL-4 binding was not complete. These results agree with
previous observations in which IL-13 was found to compete with IL-4
binding on TF-1 cells (Zurawski et al., EMBO J. 12: 2663-2670
(1993)). However, in that report the converse experiment was not
done. Here, the data show that even though IL-13 competed for IL-4
binding, IL-4 did not compete for IL-13 binding.
[0172] The competition by IL-13 for IL-4 binding sites on lymphoid
MLA 144 cells and RAJI cell lines was also investigated. These
cells were incubated with radiolabeled IL-4 with or without excess
unlabeled IL-4 or IL-13. Excess unlabeled IL-4 effectively
displaced labeled .sup.125I-IL-4 bound to MLA 144 and RAJI cells,
while excess IL-13 could not compete this binding. This observation
is at variance to that seen with RCC cells in which IL-13 competed
for IL-4 binding. The inability of IL-13 to compete for
.sup.125I-IL-4 binding to MLA 144 is consistent with the
observation that IL-13 did not bind to peripheral blood T (or MLA
144) cells.
Example 3
Subunit Structure of IL-13 and IL-4 Receptors
[0173] The subunit structure of IL-13R on RCC cells was
investigated by crosslinking studies. Cells (5.times.10.sup.6) were
labeled with .sup.125I-IL-13 or .sup.125I-IL-4 in the presence or
absence of excess IL-13 or IL-4 for 2 h at 4.degree. C. The bound
ligand was cross-linked to its receptor with disuccinimidyl
suberate (DSS) (Pierce, Rockford, Ill., USA) at a final
concentration of 2 mM for 30 min. Cells were lysed in a buffer
containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
0.02 mM leupeptin, 5.0 .mu.M trypsin inhibitor, 10 mM benzamidine
HCl, 1 mM phenanthroline iodoacetamide, 50 mM amino caproic acid,
10 .mu.g/ml pepstatin, and 10 .mu.g/ml aprotinin. The cell lysates
were cleared by boiling in buffer containing 2-mercaptoethanol and
analyzed by electrophoresis through 8% SDS/polyacrylamide gel. The
gel was subsequently dried and autoradiographed. In some
experiments, the receptor/ligand complex was immunoprecipitated
from the lysate overnight at 4.degree. C. by incubating with
protein A sepharose beads that had been pre-incubated with P7 anti
hIL-4R or anti-.gamma..sub.c antibody and analyzed as above.
[0174] The labeled .sup.125I-IL-13 cross-linked to one major
protein on all four RCC cell lines and the complex migrated as a
single broad band ranging between 68 and 80 kDa. A single band was
also observed on human pre-myeloid TF-1.J61 cells only after much
longer exposure of the gel. After subtracting the molecular mass of
IL-13 (12 kDa), the size of IL-13 binding protein was estimated at
56 to 68 kDa. The .sup.125I-IL-13 cross-linked band was not
observed when the crosslinking was performed in the presence of
200-fold molar excess of IL-13. In addition to the major band, a
faint band of approximately 45 kDa was also observed in HL-RCC and
PM-RCC but not on MA-RCC cells. This band appeared to be
specifically associated with IL-13R because unlabeled IL-13
competed for the binding of .sup.125I-IL-13. This band could
represent an IL-13R associated protein or a proteolytic fragment of
the larger band. In contrast to the displacement of .sup.125I-IL-13
binding by unlabeled IL-13, an excess of unlabeled IL-4 did not
prevent the appearance of IL-13R band in RCC cell lines. IL-13 on
the other hand competed for .sup.125I-IL-4 binding to both major
proteins on WS-RCC cells. It is of interest that
.sup.125I-IL-13-cross-linked protein was slightly larger in size in
TF-1.J61, WS-RCC, PM-RCC, and HL-RCC cell lines compared to that
seen in MA-RCC. Post-translational modifications, such as
glycosylation or phosphorylation, may account for this
difference.
Example 4
Construction of an IL-13-PE Fusion Protein
[0175] 1) Construction of a Plasmid Encoding IL-13-PE38QQR
[0176] To construct the chimeric toxin a coding region of the human
interleukin 13 (hIL-13) gene (plasmid JFE14-SR.alpha.) (Minty et
al., Nature, 362: 248 (1993), McKenzie et al. Proc. Natl. Acad.
Sci. USA, 90: 3735 (1987)) was fused to a gene encoding PE38QQR, a
mutated form of PE, thereby producing a construct (phuIL-13-Tx)
encoding the chimeric molecule. Specifically, a DNA encoding human
IL-13 was PCR-amplified from plasmid JFE14-SR.alpha.. New sites
were introduced for the restriction endonucleases NdeI and Hind III
at the 5' and 3' ends of the hIL-13 gene, respectively by PCR using
a sense primer that incorporated the NdeI site and an antisense
primer that incorporated the HindIII site.
[0177] The NdeI/HindIII fragment containing encoding hIL-13 was
subcloned into a vector obtained by digestion of plasmid
pWDMH4-38QQR (Debinski et al. Int. J. Cancer 58: 744-748 (1994)) or
plasmid pSGC242FdN1 (Debinski et al. Clin. Res. 42: 251A, (abstr.)
(1994) with NdeI and HindIII, to produce plasmid phuIL-13-Tx. The
5' end of the gene fusion was sequenced and showed the correct DNA
of hIL-13.
[0178] Human interleukin 4 (hIL-4) was cloned into an expression
vector in a similar way to hIL-13 using plasmid pWDMH4 (Debinski et
al. J. Biol. Chem. 268: 14065-14070 (1993)) as a template for PCR
amplification. Recombinant proteins were expressed in E. coli BL21
(.lambda.DE3) under control of the T7 late promoter (Id.). In
addition to the T7 bacteriophage late promoter, the plasmids also
carried a 17 transcription terminator at the end of the open
reading frame of the protein, an f1 origin of replication and gene
for ampicillin resistance (Debinski et al. J. Clin. Invest. 90:
405-411 (1992)). The plasmids were amplified in E. coli (HB101 or
DH5.alpha. high efficiency transformation) (BRL) and DNA was
extracted using Qiagen kits (Chatsworth, Calif., USA).
[0179] 2) Expression and Purification of Recombinant Proteins.
[0180] E. coli BL21 (.lambda.DE3) cells were transformed with
plasmids of interest and cultured in 1.0 liter of Super broth.
Expressed recombinant human IL-13 and human IL-13-PE38QQR were
localized in inclusion bodies. The recombinant proteins were
isolated from the inclusion bodies as described by Debinski et al.,
J. Biol. Chem. 268: 14065-14070 (1993). After dialysis, the
renatured protein of human IL-13-PE38QQR was purified on
Q-Sepharose Fast Flow and by size exclusion chromatography on
Sephacryl S-200HR (Pharmacia, Piscataway, N.J., USA) The initial
step of hIL-13 or hIL-4 purification was conducted on SP-Sepharose
Fast Flow (Pharmacia).
[0181] Protein concentration was determined by the Bradford assay
(Pierce "Plus", Rockford, Ill., USA) using BSA as a standard.
[0182] Human IL-13 and IL-13-PE38QQR were expressed at high levels
in bacteria as seen in SDS-PAGE analysis of the total cell extract.
After initial purification on SP-Sepharose (hIL-13) or Q-Sepharose
(hIL-13-PE38QQR) the renatured recombinant proteins were applied
onto a Sephacryl S-200 HR Pharmacia column. Human IL-13 and
hIL-13-PE38QQR appeared as single entities demonstrating the very
high purity of the final products. The chimeric toxin migrated
within somewhat lower than expected for 50 kDa protein M.sub.r
range which may be related to the hydrophobicity of the molecule.
The biologic activity of the rhIL-13 was exactly the same as
commercially obtained hIL-13.
Example 5
The Activity of an IL-13-PE Fusion Protein on Human Carcinoma
Cells
[0183] 3) Cytotoxic Activity of hIL-13-PE38QQR
[0184] The cytotoxic activity of chimeric toxins, such as
hIL-13-PE38QQR, were tested by measuring inhibition of protein
synthesis. Protein synthesis was assayed by plating about
1.times.10.sup.4 cells per in a 24-well tissue culture plate in 1
ml of medium. Various concentrations of the chimeric toxins were
added 20-28 h following cell plating. After 20 h incubation with
chimeric toxins, [.sup.3H]-leucine was added to cells for 4 h, and
the cell-associated radioactivity was measured. For blocking
studies, rhIL-2, 4 or 13 was added to cells for 30 min before the
chimeric toxin addition. Data were obtained from the average of
duplicates and the assays were repeated several times.
[0185] Several established cancer cell lines were tested to
determine if hIL-13-PE38QQR is cytotoxic to them. In particular,
cancers derived from colon, skin and stomach were examined. The
cancer cells were sensitive to hIL-13-PE38QQR with ID.sub.50s
ranging from less than 1 ng/ml to 300 ng/ml (20 pM to 6.0 nM)
(ID.sub.50 indicates the concentration of the chimeric toxin at
which the protein synthesis fell by 50% when compared to the
sham-treated cells). A colon adenocarcinoma cell line, Colo201, was
very responsive with an IC.sub.50 of 1 ng/ml. A431 epidermoid
carcinoma cells were also very sensitive to the action of
hIL-13-toxin; the ID.sub.50 for hIL-13-PE38QQR ranged from 6 to 10
ng/ml. A gastric carcinoma CRL1739 cell line responded moderately
to the hIL-13-toxin with an ID.sub.50 of 50 ng/ml. Another colon
carcinoma cell line, Colo205, had a poorer response with an
ID.sub.50 of 300 ng/ml.
[0186] The cytotoxic action of hIL-13-PE38QQR was specific as it
was blocked by a 10-fold excess of hIL-13 on all cells. These data
suggest that a spectrum of human cancer cells possess hIL-13
binding sites and such cells are sensitive to hIL-13-PE38QQR
chimeric toxin.
[0187] Because the hIL-13R has been suggested to share the
.gamma..sub.c subunit of the IL-2R (Russell et al. Science 262:
1880-1883 (1993)), the specificity of hIL-13-PE38QQR action on A431
and CRL1739 cells, the two cell lines with different sensitivities
to the chimeric toxin was further explored. The cells were treated
with hIL-13-PE38QQR with or without rhIL-2 at a concentration of
1.0 .mu.g/ml or 10 .mu.g/ml. The rhIL-2 did not have any blocking
action on hIL-13-PE38QQR on the two cell lines, even at 10,000 fold
molar excess over the chimeric toxin. These results indicate that
the cell killing by the hIL-13-toxin is independent of the presence
of hIL-2.
[0188] 4) IL-4, Unlike IL-2, Blocks the Action of IL-13-PE38QQR
[0189] Native hIL-4 was added to cells which were then treated with
hIL-13-PE38QQR. Unexpectedly, it was found that hIL-4 inhibited the
cytotoxic activity of the hIL-13-toxin. This phenomenon was seen on
all the tested cell lines, including Colo201, A431 and CRL1739. To
investigate the possibility that hIL-13 and hIL-4 may compete for
the same binding site, the cells were also treated with the
hIL-4-based recombinant toxin, hIL-4-PE38QQR (Debinski et al. Int.
J. Cancer 8: 744-748 (1994)). The cytotoxic action of hIL-4-PE38QQR
had already been shown to be blocked by an excess of hIL-4 but not
of hIL-2 (Id.). In the present experiment hIL-13 potently blocked
the cytotoxic activity of hIL-4-PE38QQR. Also, the action of
another hIL-4-based chimeric toxin, hIL-4-PE4E (Debinski et al. J.
Biol. Chem. 268: 14065-14070 (1993)), was blocked by an excess of
hIL-13 on Colo201 and A431 cells. Thus, the cytotoxicity of
hIL-13-PE38QQR is blocked by an excess of hIL-13 or hIL-4, and the
cytotoxic action of hIL-4-PE38QQR is also blocked by the same two
growth factors. However, IL-2 does not block the action of either
chimeric toxin. These results strongly suggest that hIL-4 and
hIL-13 have affinities for a common binding site.
[0190] This conclusion was supported by the observation of one
cytokine blocking the effect of a mixture of the two chimeric
toxins. When A431 cells were incubated with both hIL-3- and
hIL-4-PE38QQR chimeric toxins concomitantly the cytotoxic action
was preserved and additive effect was observed as expected. An
excess of hIL-13 efficiently blocked the action of a mixture of the
two chimeric toxins. Moreover, neither hIL-13 nor hIL-4 blocked
cell killing by another mixture composed of hIL-13-PE38QQR and
TGF.alpha.-PE40, a chimeric toxin which targets the EGFR
(TGF.alpha.-based chimeric toxin, TGF.alpha.-PE40) (Siegall et al.
FASEB J. 3, 2647-2652 (1992)). The same was observed on Colo201
cells.
[0191] 5) Reciprocal Blocking of Chimeric Toxins by IL-13 and IL-4
is due to Competition for Binding Sites.
[0192] The binding ability of human IL-13 was compared to human
IL-4-PE38QQR in competitive binding assays. Recombinant
hIL-4-PE38QQR was labeled with .sup.125I using the lactoperoxidase
method as described by Debinski et al., J. Clin. Invest. 90,
405-411 (1992). Binding assays were performed by a standard
saturation and displacement curves analysis. A431 epidermoid
carcinoma cells were seeded at 10.sup.5 cells per well in a 24-well
tissue culture plates at 24 h before the experiment. The plates
were placed on ice and cells were washed with ice-cold PBS without
Ca++, Mg++ in 0.2% BSA, as described (Id.). Increasing
concentrations of hIL-13 or hIL-4-PE38QQR were added to cells and
incubated 30 min prior to the addition of fixed amount of
.sup.125I-hIL-4-PE38QQR (specific activity 6.2 .mu.Ci/.mu.g
protein) for 2 to 3 hours. After incubation, the cells were washed
twice and lysed with 0.1 N NaOH, and the radioactivity was counted
in a .gamma.-counter.
[0193] Human IL-4-PE38QQR competed for the binding of
.sup.125I-hIL-4-PE38QQR to A431 cells with an apparent ID.sub.50 of
4.times.10.sup.-8 M. In addition, hIL-13 also competed for the
.sup.125I-hIL-4-PE38QQR binding site with a comparable potency to
that exhibited by the chimeric protein. More extensive binding
studies have shown that hIL-13 also competes for hIL-4 binding
sites on human renal carcinoma cell lines.
[0194] The possibility of an influence of hIL-13 or hIL-4 on the
process of receptor-mediated endocytosis and post-binding PE
cellular toxicity steps was excluded by adding to cells: (i) native
PE (PE binds to the .alpha..sub.2-macroglobulin receptor), (ii)
TGF.alpha.-PE40, and (iii) a recombinant immunotoxin
C242rF(ab')-PE38QQR (Debinski et al. Clin. Res. 42, 251A, (Abstr.)
(1994)). C242rF(ab')-PE38QQR binds a tumor-associated antigen that
is a sialylated glycoprotein (Debinski et al. J. Clin. Invest. 90:
405-411 (1992)). The expected cytotoxic actions of these
recombinant toxins were observed and neither hIL-13 nor hIL-4
blocked these actions on A431 and Colo205 cells.
[0195] 6) hIL-4 and hIL-13 Compete for a Common Binding Site on
Carcinoma Cell but Evoke Different Biological Effects
[0196] Even though hIL-13 and hIL-4 compete for a common binding
site, they induce different cellular effects. Protein synthesis was
inhibited in A431 epidermoid carcinoma cells in a dose-dependent
manner by hIL-4 alone, or by a ADP-ribosylation deficient chimeric
toxin containing hIL-4 (Debinski et al., Int. J. Cancer 58: 744-748
(1994)). This effect of hIL-4 or enzymatically deficient chimeric
toxin can be best seen with a prolonged time of incubation
(.gtoreq.24 h) and requires concentrations of hIL-4 many fold
higher than that of the active chimeric toxin in order to cause a
substantial decrease in tritium incorporation. However, when A431
cells were treated with various concentrations of hIL-13, no
inhibition (or stimulation) of protein synthesis was observed, even
at concentrations as high as 10 .mu.g/ml of hIL-13 for a 72 h
incubation. The same lack of response to hIL-13 was found on renal
cell carcinoma cells PM-RCC. Thus, while hIL-13 and hIL-4 may
possess a common binding site, they appear to transduce differently
in carcinoma cells expressing this common site, such as A431 and
PM-RCC cells.
Example 6
IL-13 Inhibits Growth of Human Renal Cell Carcinoma Cells
Independently of the P140 IL-4 Receptor Chain
[0197] Since human renal cell carcinoma cells (RCC) express a large
number of intermediate to high affinity IL-13 receptors, the effect
of IL-13 on in vitro growth of RCC cells was determined. The
interaction between the IL-13 receptor and the IL-4 receptor was
evaluated by examining the effect of anti-IL-4 and anti-IL-4R
antibodies on IL-13 binding to RCC cells and the IL-13 modulation
of RCC cell proliferation.
[0198] 1) Inhibition of RCC Cell Growth by IL-13.
[0199] Renal cell carcinoma cells--WS-RCC and PM-RCC were derived
as described previously (Obiri et al., J. Clin. Invest., supra) and
maintained in culture medium (CM) consisting of DMEM with 4.5 g/L
glucose supplemented with 10% fetal bovine serum (FBS), glutamine
(2 mM), HEPES buffer (10 mM), penicillin (100 U/ml) and
streptomycin (100 .mu.g/ml).
[0200] For proliferation assays, RCC cells were harvested, washed
and resuspended in CM in which the FBS content was reduced to 0.5%.
Ten thousand cells were plated in each well of a 96-well microtiter
tissue culture plate and cultured overnight at 37.degree. C. in a
5% CO.sub.2 environment. IL-13 and/or IL-4 (0-1000 ng/ml) were
added and incubation continued for an additional 72 h. Some
cultures were concurrently treated with anti-IL-4 or anti-IL-4R
antibody (1-10 .mu.g/ml). [.sup.3H]-thymidine (1 .mu.Ci/well) was
added for the final 20 h of incubation. At the end of the
incubation, cells were detached with trypsin or by a rapid
freeze/thaw cycle and harvested unto a glass fiber filter-mat with
a cell harvester (Skatron, Lier, Norway). [.sup.3H]-thymidine
uptake was determined with a Betaplate scintilation counter (LKB,
Gaithersburg, Md.).
[0201] IL-13 inhibited cellular proliferation by up to 50% in a
concentration dependent manner in WS-RCC and PM-RCC cell lines. The
PM-RCC cell line was more sensitive to IL-13 since 0.1-1 ng/ml
IL-13 caused a maximum inhibitory effect. The other cell line,
WS-RCC required as much as 100 ng/ml of IL-13 for maximum effect.
In addition, IL-13 at concentration of 10 ng/ml reduced
proliferation of HL-RCC cells by 33%. Higher concentrations of
IL-13 (up to 2000 ng/ml) did not have additional growth inhibitory
effect. This growth inhibitory effect of IL-13 is similar to that
observed with IL-4 on human RCC cells.
[0202] In order to examine the effect of IL-13 on the viability of
RCC cells, the cells were cultured with IL-13 (0-100 ng/ml) at
5.times.10.sup.4/MI in 12-well tissue culture plates. After 72 h,
the cells were harvested with trypsin/versene, washed and diluted
in trypan blue for cell counts. Viability was determined by trypan
blue exclusion. In control cultures, the viability (mean.+-.SD of
quadruplicate samples) was 95.+-.10% while the viability in
cultures treated with 10 or 100 ng/ml IL-13 was 92.5.+-.9.6 and
93.+-.8.9 respectively. Thus, IL-13 did not have direct cytotoxic
effect on RCC cells.
[0203] Since IL-13 competes for IL-4 binding and a mutated form of
IL-4 inhibited IL-13 and IL-4 effects (Zurawski et al., EMBO J.,
12: 2663 (1993))), the ability of anti-IL-4 or anti-IL-4R antibody
to block both IL-4 and IL-13 growth inhibitory effects was
determined. For this experiment, WS-RCC cells were treated with
IL-13 or IL-4 alone, or in the presence of a neutralizing
polyclonal antibody to hIL-4 or a monoclonal antibody to IL-4R
(M57). This approach was chosen because a suitable anti-hIL-13 was
not readily available.
[0204] [.sup.2H]-thymidine uptake was significantly inhibited
(p<0.05) in IL-13-treated cultures (1913.+-.364 cpm in treated
vs 3222.+-.458 cpm in control) and in IL-4 treated cultures
(2262.+-.210 cpm in treated vs 3222.+-.458 cpm in control). While
the IL-4-mediated inhibition of proliferation was abrogated by a
polyclonal anti-IL-4 antibody, the inhibitory effect of IL-13 was
not affected by the addition of anti-IL-4 antibody. Furthermore,
the anti-proliferative effect of IL-4 was also abrogated by M57, a
monoclonal antibody against IL-4R, but the antiproliferative effect
of IL-13 was not affected by this antibody.
[0205] When WS-RCC cells were treated with a combination of IL-4
and IL-13, the resulting inhibition of cellular proliferation was
not significantly different from that seen in cultures treated with
either cytokine alone. Thus, although IL-4 and IL-13 exert a
similar effect on RCC cell growth, their actions could not be
potentiated by using the two cytokines together.
[0206] 2) Inhibition of RCC Colony Formation by IL-13.
[0207] To confirm the observed IL-13 mediated inhibition of RCC
tumor cell proliferation, a colony formation assay was used to
evaluate the effect of IL-13 on RCC cell growth. Five hundred RCC
cells were plated in triplicate 100 cm.sup.2 tissue culture-treated
petri dishes and treated with various concentrations of IL-13. For
comparative purposes, RCC cells were also similarly, treated with
IL-4. After a 10-day culture period, the percentages of colonies
formed in control and cytokine treated groups were compared.
[0208] IL-13 inhibited colony formation in PM and WS RCC cells in a
concentration dependent manner. A maximum of 34% reduction in
colony formation was observed in WS-RCC cells. In repeated
experiments, the maximum inhibition observed in PM-RCC cells ranged
from 13-32%. The kinetics of the inhibition of colony formation in
WS-RCC cells was similar to that observed in PM-RCC cells. By
comparison, IL-4 inhibited colony formation in both cell lines to
the same extent as did IL-13. However, PM-RCC cells appeared to be
slightly more sensitive to the IL-4 effect than WS-RCC cells.
[0209] 3) Effect of Anti-IL-4 Antibody on IL-13 Binding.
[0210] As explained above, on human RCC cells, IL-13 compete for
the binding of .sup.125I-IL-4 but IL-4 does not compete for the
binding of .sup.125I-IL-13. In order to understand the mechanism
underlying the inhibition of IL-4 binding by IL-13 and to evaluate
the fidelity of ligand binding by IL-13R, the effect of anti-IL-4R
antibody on .sup.125I-IL-13 binding to PM-RCC cells, which express
both IL-4R and IL-13R, was examined. As a control, the effect of
this antibody on .sup.125I-IL-4 binding to PM-RCC cells was also
tested.
[0211] Recombinant human IL-4 and IL-13 were labeled with .sup.125I
(Amersham Corp.) by using the IODO-GEN reagent (Pierce Chem. Co.)
according to the manufacturer's instructions. Specific activity
ranged from 20 to 80 .mu.Ci/.mu.g for .sup.125IL-4 and 80 to 120
.mu.Ci/.mu.g for .sup.125IL-13. About 1.times.10.sup.6 cells were
incubated with radio labeled ligand (0.64 nM) in a buffered medium
alone or in the presence of excess cytokine (128 nM); monoclonal
(M57) or polyclonal (P2, P3, P7) rabbit antibodies raised against
human IL-4R. The antibodies were used at a final dilution of 1:64.
The incubation was done at 4.degree. C. for 2 h in a shaking water
bath. Cell bound radio-ligand was separated from free by
centrifugation through an oil gradient and bound radioactivity
determined in a gamma counter.
[0212] Both .sup.125I-IL-13 and .sup.125I-IL-4 specifically bound
to PM-RCC cells (181,650.+-.3,182 cpm and 9,263.+-.576 cpm
respectively). Unlabeled IL-13 competed well for .sup.125I-IL-13
binding, however, neither IL-4 nor any of three different
polyclonal antibodies to IL-4R competed for the binding of
.sup.125I-IL-13 on PM-RCC cells. Similarly, a monoclonal antibody
to IL-4R (M57) did not black the binding of .sup.125I-IL-13 to
PM-RCC cells. In contrast, IL-4, IL-13 and anti-IL-4R antibody (P7)
all competed for .sup.125I-IL-4 binding on these cells.
[0213] This Example demonstrates that IL-13 inhibits the
proliferation of human RCC cells in a concentration dependent
manner. A maximum of 50% growth inhibition was observed and this
growth inhibitory effect of IL-13 was supported by the results of a
colony formation assay. It is noteworthy that the same
concentration range of IL-13 inhibited colony formation in both RCC
cell lines. Although a similar magnitude of growth inhibition has
been reported for IL-4, this is the first report of a direct
anti-tumor effect of IL-13 on RCC cells. Furthermore, inhibitory
effects of IL-4 on colony formation in RCC cells have not been
previously reported.
[0214] The antitumor effects of IL-13 were independent of IL-4 and
did not involve IL-4R. This is evidenced by the fact that
polyclonal or monoclonal antibodies to IL-4 or to the 140 kDa
subunit of IL-4R had no effect on the growth inhibitory effect of
IL-13. As was previously observed with IL-4, the inhibitory effect
of IL-13 on RCC growth was cytostatic rather than cytotoxic since
the viability in cells cultured with 10 or 100 ng/ml IL-13 was
similar to that observed in control cultures after 72 h
treatment.
[0215] Recently, IL-13 was shown to directly inhibit the
proliferation of normal and leukemic B precursor cells in vitro by
30% (Renard et al., Blood, 84: 2253-(1994)). This growth inhibitory
effect of IL-13 was abrogated by an antibody to the 140 kDa subunit
of IL-4R. Similarly, the growth stimulatory effect of IL-13 on TF-1
cells was also shown to be blocked by an antibody to IL-4R (e.g.,
Tony et al., Europ. J. Biochem., 225: 659 (1994)). However, in this
study, none of 3 different antibodies to IL-4R blocked the growth
inhibitory effect of IL-13. These contrasting findings may suggest
that the antibodies used in this study and those used by others are
directed at different epitopes on the IL-4R protein. An alternative
explanation, which we favor, is that IL-13R on RCC are structurally
different from those expressed on lymphoid cells.
[0216] Structural differences between IL-4R expressed on RCC and
those expressed on lymphoid cells have been identified. These
include the absence of the common gamma chain of the receptors for
IL-2, 4, 7, 9, and 15 in tumor cell IL-4R, although this chain is
present in IL-4R of immune cells (Obiri et al. Oncol. Res., 6: 419
(1994)).
[0217] Previous studies have demonstrated that antibodies to IL-4R
block cellular responsiveness to IL-13 (Tony et al., Europ. J.
Biochem., 225: 659 (1994)). However, the effect of these antibodies
on the binding of .sup.125I-IL-13 to the cells was not
investigated. We report here that the binding of radio-labeled
IL-13 to its receptors on RCC cells could not be blocked by a
polyclonal antibody to IL-4R which did block the binding of
radio-labeled IL-4 to its receptors. These data suggest that in RCC
cells, IL-13 interaction with its receptor does not involve the 140
kDa subunit of-IL-4R and IL-13 effects are probably mediated by
receptors that are not shared with IL-4.
[0218] Nevertheless, results from the above described Examples do
suggest some common element(s) between IL-4R and IL-13R. For
example, IL-13 binds to a .sup.-70 kDa protein and competes for
IL-4 binding but IL-4 did does compete for IL-13 binding in RCC
cells. In addition, IL-4 cross links to a .sup.-70 kDa protein in
addition to its primary 140 kDa binding protein. Taken together,
these data suggest that the -70 kDa protein binds both IL-13 and
IL-4. This indicates that the -70 kDa protein may be a homodimer in
which one of the constituents binds IL-13 alone while the other
binds both IL-13 and IL-4. The data further suggest that because it
binds to both putative components of the .sup.-70 kDa protein,
IL-13 has a higher binding affinity to this protein than does IL-4
which appears to bind, at most, one component of the IL-13
receptor. Such an arrangement explains the finding that IL-13
competes for .sup.125I-IL-4 binding while IL-4 does not compete for
.sup.125I-IL-13 binding on these cells. Finally, since antibody to
IL-4R did not block IL-13 binding, and .sup.125I-IL-13 cross
linking to the p140 form of the IL-4R was not detected, in RCC
cells, IL-13 does not appear to utilize the 140 kDa IL-4 binding
subunit.
[0219] The observation that the combination of IL-4 and IL-13 does
not inhibit RCC cell proliferation any better than either cytokine
alone suggests that the anti-proliferative effects of IL-4 and
IL-13 are mediated through a common receptor subunit or common
signaling pathway. This is consistent with the notion of a shared
receptor or receptor component for the two cytokines and the
observation that both IL-13 and IL-4 phosphorylate a member of the
Janus family of kinases (JAK 1) as well as the 140 kDa subunit of
IL-4R and activate the same signal transducer and activator of
transcription (STAT 6) proteins in different cell types.
[0220] In summary, IL-13, like IL-4 directly inhibits RCC
proliferation in vitro. The IL-13 effect is independent of IL-4
since anti-IL-4R antibody did not inhibit IL-13 binding to its
receptor and anti-IL-4R antibody did not inhibit the IL-13 effect
on RCC cells. These findings suggest that IL-13R directed chimeric
molecules are particularly useful for the management of RCC.
Example 7
Targeting of Interleukin-13 Receptor on Human Renal Cell Carcinoma
Cells by Recombinant IL-13-PE Cytotoxins
[0221] 1) Cytotoxicity of IL-13-Toxin Fusion Protein.
[0222] The cytotoxic activity of IL-4-toxins was tested as
described above. Typically, 10.sup.4 RCC tumor cells or other cells
were cultured in leucine-free medium with or without various
concentrations of IL-toxin for 20-22 hours at 37.degree. C. Then 1
.mu.Ci of [.sup.3H]-Leucine (NEN Research Products, Wilmington,
Deleware, USA) was added to each well and incubated for an
additional 4 hours. Cells were harvested and radioactivity
incorporated into cells was measured by a Beta plate counter
(Wallac-LKB, Gaithersburg, Md., USA).
[0223] Four primary cell cultures (PM-RCC, WS-RCC, MA-RCC &
HL-RCC) and 1 long term culture (RC-2) of RCC cell lines were
tested because of the large number of IL-13 receptors expressed by
human RCC cells (see Example 1). RCC cells were sensitive to the
cytotoxic activity of IL13-toxin with IC.sub.50 ranging from as low
as 0.03 ng/ml to 350 ng/ml (<2 fM to 1 nM) (Table 2). All four
primary cultures of RCC cells generated in our laboratory (18)
seemed to be more sensitive to IL13-PE38QQR compared to long term
RCC cell line (CAKI-1). The cytotoxic activity of IL13-toxin was
specific and mediated through IL-13R, because excess IL-13
neutralized the cytotoxic activity of IL13-toxin. Thus, RCC cells
are killed by IL13-PE38QQR at uniquely low concentrations of the
chimeric protein.
2TABLE 2 Cytotoxic activity of IL13-PE38QQR on human RCC tumor cell
lines. IC.sub.50 (ng/ml).sup.a IL-13 binding Reference Tumors mean
.+-. SD sites/cell No. HL-RCC 0.03, <0.1 150,000 13 PM-RCC 0.090
.+-. 0.01 26,500 13 MA-RCC 0.340 .+-. .15 5,000 13 WS-RCC 17.500
.+-. 3.50 2,000 13 CAKI-1 350.000.sup.b <100 --.sup.c
IL13-PE38QQR (474 amino acid protein) is composed of IL-13 (114
N-terminal amino acids) and domain II and domain III of PE molecule
(Debinski et al., J. Biol. Chem., 270: 16775 (1995)).
.sup.aIC.sub.50, the concentration of IL13-toxin at which 50%
inhibition of protein synthesis is observed compared to untreated
cells and was determined as described under "methods". The mean
IC.sub.50 for individual tumors is shown and was determined from
2-5 experiments for four RCC tumor cell lines. .sup.bSingle
experiment performed in quadruplicate using 5 different
concentration of IL13-toxin. .sup.ccurrent data
[0224] 1) Correlation Between IL-13R Expression and Sensitivity to
IL13-Toxin.
[0225] As described above, the primary RCC cell lines, such as
PM-RCC, WS-RCC, HL-RCC, and MA-RCC expressed varied numbers of
high- to intermediate-affinity IL-13R. However, IL-13 binding
characteristics on CAKI-I RCC cell line was not determined. IL-13
binding studies were therefore performed on these RCC cells
utilizing [.sup.125I]-IL-13.
[0226] IL-13 was iodinated with IODOGEN reagent (Pierce, Rockford,
Ill., USA) according to manufacturer's instructions. The specific
activity of radio-labeled IL-13 ranged between 44 to 128
.mu.Ci/.mu.g. The IL-13 binding assay was performed by as described
above (see Example 1). Briefly, RCC tumor cells were harvested
after brief incubation with versene (Biowhittaker), washed three
times in Hanks balanced salt solution and resuspended in binding
buffer (RPMI 1640 plus 1 mM HEPES and 0.2% human serum albumin).
For IL-13 displacement assay, RCC (1.times.10.sup.6/100 .mu.l)
cells were incubated at 4.degree. C. with .sup.125I-IL-13 (100-200
pM) with or without increasing concentrations of unlabeled IL-13 or
IL13-PE38QQR. Following a 2 h incubation, cell bound radio-ligand
was separated from unbound by centrifugation through a phthalate
oil gradient and radioactivity determined with a gamma counter
(Wallac).
[0227] CAKI-1 RCC cell line did not bind radiolabeled IL-13 well
and only expressed <100 IL-13 binding sites/cell (Table 1). The
sensitivity of these cell lines to IL13-toxin also varied depending
on the number of IL-13 binding sites per cell. CAKI-1 RCC cell line
expressed the least number of IL-13 binding sites and were least
sensitive to IL13-toxin. In contrast, HL-RCC cells were extremely
sensitive and expressed 150,000 IL-13 binding sites/cell.
[0228] 2) In vivo Passage of MA-RCC does not Decrease Sensitivity
to IL3-Toxin.
[0229] In order to determine the antitumor activity of IL13-toxin
against human RCC, human RCC cells were grown as subcutaneous
tumors in nude mice, irradiated (300 rads) nude mice and in SCID
mice. However, these RCC cells did not grow consistently in any of
these immunoincompetent mice. In some cases tumors did grow very
slowly but became centrally necrotic with a white rim of viable RCC
cells.
[0230] Therefore, antitumor activity of IL13 toxin was not
evaluated in vivo. However, MA-RCC were passaged in nude mice and
the passaged tumors were used to prepare single cell suspensions.
These cells did grow in tissue culture and after 1-3 passages,
their sensitivity to IL13-toxin was determined.
[0231] MA-RCC were very sensitive to IL13-toxin and passaging of
these RCC cells in vivo twice did not decrease their sensitivity.
These data suggest that IL-13R levels do not change by in vivo
passaging of RCC tumor cells.
[0232] 3) IL13-Toxin is not Cytotoxic to Immune Cells, Monocytes,
Bone Marrow-Derived Cells, and Burkitt's Lymphoma Cells.
[0233] The cytotoxic activity of IL13-PE38QQR was also examined on
PHA-activated T cells, a CD4+ T cell lymphoma line (H9), normal
bone marrow cells, EBV-transformed B cell line, 2 Burkitt's
lymphoma cell lines and a premonocytic cell line (U937). As shown
above in Example 1, PHA-activated T cells, H9 cells, and U937 cells
did not express detectable numbers of IL-13R. Consistent with these
observations, IL13-PE38QQR was not cytotoxic to any of these cell
types. EBV-transformed B cell line did express about 300
IL13-binding sites/cell, however, IL13-toxin was not cytotoxic to
them. Although IL-13R expression was not tested on human bone
marrow cells or Burkitt's lymphoma cell lines; based on their
insensitivity to IL13-toxin, it is expected that these cells also
do not express IL-13R or express a low number of these
receptors.
[0234] 4) Clonogenic Assay.
[0235] The antitumor activity of IL13-PE38QQR was also tested by a
colony-forming assay. Five hundred PM-RCC cells were plated in 100
mm petri dishes and the next day triplicate plates received IL-13
(20 ng/ml), IL13-PE38QQR (50 ng/ml) or control medium. The cells
were cultured for 10 days at 37.degree. C. in a CO.sub.2 incubator.
Media was then removed and colonies were fixed and stained with
0.25% crystal violet in alcohol. Colonies containing 50 or more
cells were scored. The surviving fraction was calculated as the
ratio of the number of colonies formed in treated and untreated
cells and presented as percent survival.
[0236] Human PM-RCC cells formed colonies when 500 cells were
cultured in petri dishes. Using this number of cells, PM-RCC cells
formed 175 colonies with a clonogenic efficiency of 35%. When these
cells were treated with IL13-PE38QQR for 10 days, only 32 colonies
were formed (Table 3). However, 123 or 175 colonies were formed
when cells were treated with recombinant IL-13 or media alone
respectively.
3TABLE 3 Effects of IL-13 and IL-13-PE38QQR on PM-RCC cells by
clonogenic assay. % Surviving No. Colonies .+-. SD fraction PM RCC:
Control 175 .+-. 5 100 IL13-PE38QQR 32 .+-. 4 18 IL-13 123 .+-. 3
70 HL RCC: Control 348 .+-. 9 100 IL13-PE38QQR (5 ng/ml) 4 .+-. 0.8
1 IL13-PE38QQR (15 ng/ml) 1 .+-. 1 0.3 IL-13 232 .+-. 12 67
[0237] 5) IL-4 does not Block the Cytotoxic Activity of
IL13-PE38QQR on RCC Cells.
[0238] IL-13 competed for the binding sites of IL-4 while IL-4 did
not compete for the binding site of IL-13. However, in other cancer
cell types IL-4 neutralized the cytotoxicity mediated by
IL13-PE38QQR. The ability of IL-4 to neutralize the cytotoxicity of
IL13-toxin on RCC cells was therefore tested. Only IL-13 blocked
the cytotoxicity of IL13-toxin, while IL-4 did not block this
cytotoxicity in all three RCC cell lines tested.
[0239] 6) Binding Affinity of IL13-Toxin on Human RCC Cells.
[0240] The binding affinity of IL13-PE38QQR to IL-13R was then
examined. HL-RCC or PM-RCC cells were utilized for this purpose.
These cells were incubated with a saturating concentration of
radiolabeled IL-13 in the absence or presence of various
concentrations of IL-13 or IL13-PE38QQR. In HL-RCC cells the
IC.sub.50 (the protein concentration at which 50% displacement of
[.sup.125]-I-IL-13 binding is observed) for native IL-13 was
.sup.-20.times.10.sup.-9 M, compared to .sup.-180.times.10.sup.-9 M
with IL13-PE38QQR. Thus IL13-toxin bound to IL-13R with about 8-10
fold lower affinity compared to IL-13.
[0241] The foregoing experiments show that an IL-13 based
cytotoxin, IL13-PE38QQR, is highly cytotoxic to human renal cell
carcinoma cells. The IC.sub.50 in RCC cell lines ranged from less
than 0.03 ng/ml to 350 ng/ml. The cytotoxicity of the IL13-toxin
was specific and mediated through IL-13R because excess IL-13
neutralized the cell killing activity of IL13-PE38QQR. These
results corroborate with the data generated in a clonogenic assay
that demonstrate a significant inhibition of colony formation by
IL13-toxin.
[0242] Resting human cells including non-activated T cell line
(H9), EBV-transformed B cell line, and promonocytic (U937) cell
lines were not sensitive to the cytotoxic effect of IL13-toxin.
Similarly, PHA-activated human T cells and cells obtained from
normal bone marrow biopsy were also insensitive to the cytotoxic
effect of IL13-PE38QQR. It has previously been reported that
hematologic progenitor cell lines and fresh human bone marrow cells
express low numbers of IL-4 receptors (e.g., Lowenthal et al. J.
Immunol., 140: 456 (1988)). However, IL-13R expression on these
cells has not been determined. A recent study reported that IL-13
has a direct regulatory role in the proliferation and
differentiation of primitive murine hematopoietic progenitor cells
(Jacobsen et al. J. Exp. Med., 180: 75 (1994)) indicating
expression of some level of IL-13R on these cells. However, the
example shows that IL13-toxin was not cytotoxic to fresh bone
marrow derived cells indicating that progenitor cells probably
express insufficient amount of IL-13R or receptors on these cells
are not susceptible to the cytotoxic action of IL13-toxin.
[0243] It was shown above that IL-13 competes for the binding of
IL-4 while IL-4 does not compete for the binding of IL-13 on RCC
cells (Example 2). Similar to these results, the data in this
example show that IL-4 does not neutralize the cytotoxic effect of
IL13-PE38QQR.
[0244] It has been previously demonstrated that IL4 based cytotoxin
(IL4-PE4E) is highly cytotoxic to human RCC cells. A comparison was
not made between IL13-PE38QQR and IL4-PE4E because the PE portion
in these two chimeric proteins is different. However, both IL-13
and IL-4 competed with the cytotoxicity of IL4-toxin. Similarly, a
mutant IL-4 protein blocked the proliferative response generated by
IL-4 and IL-13. These data suggest that the receptors for IL-13 and
IL-4 share a component.
[0245] The data on RCC cells showed that [.sup.125]-I-IL-13
crosslinked to one major protein of .sup.-70 kDa, which appeared to
be similar in size to the smaller of the two subunits of IL-4R. The
competition of IL-13 for the binding sites of IL-4, suggests that
the .sup.-70 kDa protein is shared between these two receptors.
Also, IL-4 and IL-13 compete reciprocally to an internalized
receptor form on some carcinoma cell lines. Recent data demonstrate
that both IL-4 and IL-13 caused the phosphorylation of 140 kDa 1L-4
binding protein. In addition, antibody to 140 kDa IL-4 binding
protein blocked the effects of IL-13 on B cells. While these
studies, suggest that the 140 kDa IL-4 binding protein may be
shared between these two cytokine receptors, crosslinking of
[.sup.125I]-IL-13 to the 140 kDa protein was not observed even
though [.sup.125I]-IL-4 crosslinked to this protein. These data
suggest that either the 140 kDa IL-4 binding protein does not share
a chain with IL-13R or the 140 kDa protein is a non-IL-13 binding
component of the IL-13R system which is why IL-4 does not compete
for the binding of IL-13.
[0246] It is of interest to note that IL13-toxin binds to IL-13
receptor with a lower affinity compared to that of IL-13. Since PE
molecule was attached to the C-terminus of the IL-13 molecule,
these data suggest that, similar to IL-4, IL-13 may interact with
its receptor predominantly through C-terminal end residues. In
addition, these data also suggest that a chimeric IL13 toxin
molecule in which the toxin moiety is attached at a site away from
the C-terminus residues should be more cytotoxic to cancer
cells.
[0247] In summary, these results indicate that IL13-toxin
IL13-PE38QQR is highly cytotoxic to human RCC cells which express
high numbers of IL-13R. Because resting or activated immune cells
or bone marrow cells are not sensitive to IL13-toxin, the data
indicate that this toxin is useful for the treatment of RCC without
being cytotoxic to normal immune cells.
Example 8
Human Glioma Cells Overexpress IL-13 Receptors and are Extremely
Sensitive to IL-13PE Chimeric Proteins
[0248] In order to evaluate the efficacy of the chimeric
immunotoxins of this invention on brain tumors, cytotoxicity (as
evaluated by inhibition of protein synthesis) and competitive
inhibition assays were performed on a number of brain tumor cell
lines as described below.
[0249] 1) Protein Synthesis Inhibition Assay.
[0250] The cytotoxic activity of chimeric toxins (e.g.,
hIL13-PE38QQR) was tested on brain tumor cell lines. This group of
cells is represented by human gliomas and includes U-373 MG,
DBTRG-05 MG, A-172, Hs 683, U-251 MG, T-98G, SNB-19, and SW-1088,
and also one human neuroblastoma SK-N-MC cell line. The majority of
cell lines was obtained from the ATCC and they were maintained
under conditions recommended by the ATCC. The SNB-19 cell line was
obtained from National Cancer Institute/Frederick Cancer Research
Facility, DCT tumor repository. Both SNB-19 and SW-1088 cell lines
are of neuroglial origins.
[0251] Usually about 1.times.10.sup.4 cells/well were plated in a
24-well tissue culture plate in 1 ml of medium and various
concentrations of chimeric immunotoxin were diluted in 0.1% bovine
serum albumin (BSA)/phosphate-buffered saline (PBS) and 25 .mu.l of
each dilution was added to 1 ml of cell culture medium. After 20 hr
incubation with the immunotoxins, [.sup.3H]-leucine was added to
the cells for 3-5 hr, and the cell-associated radioactivity was
measured using a beta counter.
[0252] For blocking studies (i) recombinant hIL13 (rhIL13) or (ii)
rhILA was added to cells for 20-30 min before the addition of
chimeric toxins (CTs). Data were obtained from the average of
duplicates and the assays were repeated several times.
[0253] The cancer cells were sensitive to hIL13-PE38QQR with
IC.sub.50s ranging from less than 0.1 ng/ml to more than 300 ng/ml
(2 pM to 6.0 nM). (The IC.sub.50 was calculated as the immunotoxin
toxin concentration that causes 50% inhibition of tritiated leucine
incorporation by the test cell line.) The cell lines fell into
roughly three groups according to their responsiveness to the
chimeric toxin. The first group consisting of U-373 MG, U-251 MG,
SNB-19, and A-172 was killed by hIL13-PE38QQR at the lowest
concentrations with IC.sub.50s ranging from less than 0.1 to 0.5
ng/ml (2 to 10 pM). In particular, SNB-19 and A-172 had IC.sub.50s
of about 0.05 ng/ml. The second group of glioma cell lines composed
of DBTRG MG and Hs-683 cells also responded very well to the
hIL13-toxin with IC.sub.50s in a range of 1-10 ng/ml (20-200 pM).
The third group of glioma cell lines represented by T-98G and SW
1088 had poorer responses with IC.sub.50s of 300 and >1000
ng/ml, respectively. The only human cancer cell line of neural
origin tested, the SK-N-MC neuroblastoma cell line, responded
relatively poor to the chimeric toxin.
[0254] The cytotoxic action of hIL13-PE38QQR was specific as it was
blocked by a 10- or 100-fold excess of hIL13 on the studied cells.
These data indicate that most of the human glioma cancer cells
examined possess hIL13 binding sites and such cells are extremely
sensitive to hIL13-PE38QQR.
[0255] 2) Cytotoxic Activities of Other Cytokine-Based Chimeric
Proteins in Glioma Cells.
[0256] The cytotoxic action of hIL13-PE38QQR was compared to that
of chimeric toxins containing other interleukins, such as hIL4 or
hIL6. It has already been shown that some glioma cell lines can be
killed by hIL4-PE4E with IC.sub.50s exceeding 10 ng/ml (Puri et al.
Int. J. Cancer, 58: 574-581 (1994)). hIL13-PE38QQR was cytotoxic to
U-251 MG, U-373 MG and DBTRG MG cell lines with IC.sub.50s much
below 10 ng/ml. The cytotoxin hIL4-PE38QQR, a hIL4-based chimeric
toxin resembling hIL13-PE38QQR, killed glioma cell lines, but at a
concentration ranging from a factor of 10 to almost a factor of
1000 higher than that of hIL13-based toxin.
[0257] The IC.sub.50s for hIL4-PE38QQR were higher than that seen
with the hIL4-PE4E variant of the chimeric toxin (Debinski, et al.
J. Biol. Chem., 268: 14065-14070 (1993), Puri et al. Int. J.
Cancer, 58: 574-581 (1994)) which is consistent with observations
made with other growth factor-based chimeric proteins (Siegall et
al. Cancer Res., 51: 2831-2836 (1991)). Interestingly, hIL6-PE40
was also active on some human glioma cells and its activity was
similar to that of the hIL4-toxin or better. However, hIL6-PE40 was
still less active than the hIL13-based chimeric protein. These
results show that human glioma cell lines are extremely sensitive
to hIL13-PE38QQR and the cytotoxic activity of the IL13 directed
cytotoxin is considerably better than that of other
interleukin-based chimeric toxins.
[0258] 3) Competitive Binding Assay.
[0259] The previous examples demonstrated that the action of
hIL13-PE38QQR on several solid tumor cell lines is hIL13- and
hIL4-specific, i.e., it can be blocked by these two cytokines but
not by IL2. However, it was also observed that hIL4 cannot compete
for hIL13 binding sites (Obiri et al. J. Biol. Chem., 270:
8797-8804 (1995)) and it cannot block the cytotoxic action of the
hIL13-based chimeric protein on some other cancer cell lines. Thus,
the ability of hIL4 to block the IL13-toxin cytotoxin in glial
cells was determined.
[0260] The hIL4 cytokine was ineffective in preventing the
cytotoxicity of hIL13-PE38QQR on both U-251 MG and U-373 MG cell
lines. On the other hand, hIL13 did block the cytotoxic activity of
hIL4-PE38QQR. Thus, the cytotoxicity of hIL13-PE38QQR was blocked
by an excess of hIL13 but not of hIL4, and the cytotoxic action of
hIL-PE38QQR was blocked by hIL13.
[0261] 4) Human Glioma Cell Lines Express a Number of Receptors for
IL13.
[0262] To verify that the cytotoxic activity of hIL13-PE38QQR is
specific and mediated by hIL13 receptors, competitive binding
assays were performed. Recombinant hIL13 was labeled with .sup.125I
(Amersham Corp.) by using the IODO-GEN reagent (Pierce) according
to the manufacturer's instructions, as previously described (Obiri
et al J. Biol. Chem., 270: 8797-8804 (1995)). The specific activity
of the radiolabeled cytokines was estimated to range from 20 to 100
.mu.Ci/.mu.g of protein. For binding experiments, typically
1.times.10.sup.6 tumor cells were incubated at 4.degree. C. for 2 h
with .sup.125I-hIL13 (100 pM) with or without increasing
concentrations (up to 500 nM) of unlabeled cytokine. The data were
analyzed with the LIGAND program (Munson, et al., Analy. Biochem.
107: 220-239 (1980)) to determine receptor number and binding
affinity.
[0263] Unlabeled hIL13 competed for the binding of .sup.125I-hIL13
to U-373 MG cells efficiently. The Scatchard plot analyses of
displacement experiments revealed one single binding site for hIL13
of intermediate affinity (K.sub.d=1.8 nM). There were around 16,000
binding sites for hIL13 on the U-373 MG cell line. The presence of
hIL13 receptors in other human glioma cell lines was also
evaluated. As seen in Table 4, the glioma cells had receptors for
hIL13 ranging from 500 to 30,000 molecules per cell. The hIL-13Rs
expressed in human glioma cells are of intermediate affinity with
K.sub.ds ranging from 1 to 2 nM. It is noteworthy that four out of
five cell lines studied had very
4TABLE 4 Human IL-13 binding to human glioma cells. Binding Sites*
Kd hIL-13-PE38QQR Cell Line molecules/cell (% CV) (nM) IC.sub.50
(ng/ml) A-172 22,600 (15) 1.6 <1 U-251 MG 28,000 (12) 2.1 <1
SNB-19 17,580 (19) 1.4 <1 T-98G 549 (37) 1.0 200 U-373 MG 16,400
(14) 1.8 <1 *1 .times. 10.sup.6 cells were incubated with
125I-hIL-13 (100 pM) with or without increasing concentrations (up
to 500 nM) of unlabeled hIL-13. Displacment curves and scatchard
analyses were generated from the binding data using the LIGAND
program (Munson et al., Analy. Biochem., 107: 220-239 (1980)).
[0264] high numbers of hIL-13R, i.e., above 15,000 molecules per
cell. The very same cell lines were also the most responsive to the
action of hIL-13-PE38QQR (Table 1). The T-98 G cell line was poorly
responsive to the hIL-13-toxin.sup.3 and was found to have only
around 500 hIL-13 binding sites per cell (Table 1). Thus, specific
hIL-13Rs are expressed in glioma cell lines and they mediate the
cytotoxicity of hIL-13-PE38QQR.
[0265] These experiments establish that human glioma cell lines
express large numbers of the receptor for the cytokine, IL13 and
that it is possible to target hIL-13R with a chimeric toxin
composed of the IL13 interleukin and a derivative of PE (e.g.,
PE38QQR). The hIL13-PE38QQR toxin is extremely active on several
glioma cell lines and most of these cell lines are killed at
concentrations below 1 ng/ml (<20 pM).
[0266] The action of hIL13-PE38QQR on glioma cells appears
hIL13-specific because (i) hIL13 alone blocks the cytotoxicity of
the chimeric toxin on all of the studied cell lines, and (ii) rhILA
does not prevent the cytotoxic action of hIL13-PE38QQR on U-251 MG
and U-373 MG glioma cells. The latter observation is different from
the one made on adenocarcinomas of the skin, stomach and colon
origins (Debinski et al., J. Biol. Chem., 270: 16775-16780 (1995)).
The action of IL13-PE38QQR was blocked efficiently by rhILA on
these adenocarcinoma cell lines.
[0267] Receptors for IL4 and IL13 are complex and they have some
common features detected in various systems, such as normal or
malignant human cells. However, the U-251 MG cell line does not
bind rhIL4 in a standard binding assay at 4.degree. C. while the
number of hIL13 binding sites is high on these cells. This
phenomenon most probably explains why rhIL4 does not block the
action of hIL13-PE38QQR on these cells. Thus, the receptors for
hIL13 and hIL4 in glioma cells are different from those found in
several solid tumor cell lines.
[0268] The hIL13-PE38QQR cytotoxin is considerably more active on
glioma cell lines than the comparable IL4-based chimeric toxin.
This difference in cytotoxicity is presumably due to the difference
in numbers of IL13 and IL4 molecules that can be bound by glioma
cells. Many human glioma cells bind more than 15,000 and up to
30,000 molecules of IL13 per cell while these cells bind from less
than 3,000 to very few molecules of IL4 per cell. Interestingly,
some human glioma cells can also be killed by a chimeric toxin
containing hIL6 (Siegall et al., Cancer Res., 51: 2831-2836
(1991)). However, the potency of hIL6-PE40 chimeric protein is
lower from that of hIL13-PE38QQR.
Example 9
Chimeric Toxins Having Increased Cytotoxicity
[0269] Two chimeric toxins were produced that had higher specific
toxicities than IL-13-PE38QQR. The first cytotoxin was an
IL-13-PE4E toxin where PE4E is a "full length" PE with a mutated
and inactive native binding domain where amino acids 57, 246, 247,
and 249 are all replaced by glutamates.
[0270] The second fusion protein was circularly permuted human
IL-13 (cpIL-13) fused to PE38QQR. In particular, the circularly
permuted IL-13 was produced by selecting the methionine (Met) at
position 44 of human IL-13 (hIL-13), just at the beginning of the
putative second alpha-helix of hIL-13, as the "new" N-terminal end
of the cytokine. The "old" N-, and C-termini were connected by a
short peptide having the sequence Gly-GLy-Ser-Gly. The circularly
permuted IL-13 (cphIL-13) was cloned in a way that the "new"
C-terminus of cphIL-13 (Gly-43 in a wild-type cytokine) was fused
to the N-terminal Gly of PE38QQR.
[0271] The plasmid encoding cphIL-13-PE38QQR was constructed as
follows: Plasmid phIL13, encoding the 114 amino acids of hIL13
(see, e.g., Debinski et al., J. Biol. Chem. 270: 16775-16780
(1995)) served as a template for amplification of two separate
fragments of hIL13; a fragment consisting of amino acids 1-43 and a
fragment consisting of amino acids 44-114 or hIL-13 respectively.
Both hIL13 1-43 and hIL13 44-114 were produced by PCR-amplification
using two set of primers, primers cp1/cp2 and cp3/cp4, respectively
(see Table 5, Sequence ID Nos. 1, 2, 3, and 4 respectively).
Primers cp1 and cp4 introduced a new cloning site for Bam HI
restriction endonuclease; primer cp2 encoded for a Hind III site
and primer cp3 for Nde I restriction site. PCR-amplified cDNAs
encoding for hIL13 1-43 (130 base-long) was cut with Bam HI and
Hind III enzymes, and hIL13 44-114 (210 base-long) was cut with Nde
I and Bam HI. These two fragments of DNA were ligated in a
three-fragment ligation reaction to a vector encoding hIL13-PE38QQR
(Id.) and cut with Nde I and Hind III restriction enzymes.
5TABLE 5 PCR primers used to circularly permute hIL-13. PCR
Sequence ID primer Sequence No. cp1
5'-GTGACTGCAGGTGTCCATATGTACTGTGCAGCCCTGGA-3' 1 cp2
5'-CCCAAACCGCGGGATCCACCGTTGAACCGTCCCTCGCGAA-3' 2 cp3
5'-GCAGTCGTGGGTGGATCCGGCGGTTCCCCAGGCCCTGTGCCTCC-3' 3 cp4
5'-TGGTGCAGCATCAAAAGCTTTGCCAGCTGTCAGGTTGATGC-3' 4
[0272] The resulting plasmid, pCP/hIL13-PE38QQR, carried the cDNA
for a circularly permuted hIL13 in which the new N-terminus starts
at Met 44 of the wild type interleukin-13. Four additional amino
acids (GlyGlySerGly) are located in between the residues 114 and 1
of the wild type hIL13. Circularly permuted hIL13 was linked to the
first amino acid of PE38QQR. The cphIL-13-PE38QQR was expressed in
E. coli and purified to homogeneity.
[0273] Both hIL-13-PE4E and cphIL-13-PE38QQR exhibited cytotoxic
activities that were two to ten fold better than those seen with
hIL-13-PE38QQR. IC.sub.50s were as low as <0.1 ng/ml (<2 pM)
on several glioma cell lines. Fresh human glioma explant cells were
also killed at these low concentrations. The data suggest that the
recepotr for IL-13 is an excellent target for the treatment of
human gliomas using IL-13R directed cytotoxins.
Example 10
Activity of IL-13R Directed Cytotoxins on Neural Cancers
[0274] The cytotoxicity of two chimeric toxins (hIL-13-PE38QQR and
hIL-13PE4E) was tested on cancer cell lines of neural origins. The
DAOY, TE671, and D283 medulloblastoma cell lines were all
responsive to hIL-13 fused to PE4E. The IC.sub.50s recorded in an
MTS colorimetric cytotoxicity assay were in a range of <1 ng/ml
to 50 ng/ml (<20 pM to 1 nM, respectively). In addition, human
medulloblastoma explant cells also responded well to
hIL-13-PE4E.
[0275] On the other hand, the SK-N-MC and Neuro-2A neuroblastoma
cells were poorly responsive (IC.sub.50s>4 nM). The data,
however, suggest that the overexpression of a receptor for hIL-13
is not restricted to gliomas, but it can be observed in
neuron-derived cancers.
Example 11
IL-13R Targeted Cytotoxins are Effective Against Kaposi's
Sarcoma
[0276] The recombinant immunotoxin IL-13-PE38QQR was also tested
against Kaposi's sarcoma cell lines (NCB59, KS248, KS220B, KS54A,
and ARL-13). All of the cell lines were cytotoxin sensitive with
ID.sub.50s ranging from about 8 ng/ml to about 180 ng/ml. The
Kaposi's sarcoma cell lines all expressed IL-13 receptors at higher
levels than normal cells, however the levels were lower than the
IL-13R expression levels found in renal cell carcinoma or in
gliomas.
[0277] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
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